Crystallization of IGF-1

ABSTRACT

Crystalline IGF-1 is provided along with a method for production thereof. Crystallizing IGF-1 comprises the steps of mixing an aqueous solution comprising IGF-1 with a reservoir solution comprising a precipitant to form a mixture; and crystallizing the mixture, optionally also recrystallizing and isolating the crystalline IGF-1. In addition, a method for identifying IGF-1 indirect agonists is provided using a detergent as a standard for the level of inhibition of binding of IGFBP-1 or IGFBP-3 to IGF-1 and/or using the coordinates of the binding pockets of IGF-1 to which a candidate indirect agonist binds for structure-based drug design.

RELATED APPLICATIONS

This is a divisional application 1 claiming the benefit of the priorityof application Ser. No. 10/066,009 filed on Feb. 1, 2002 now U.S. Pat.No. 7,084,240 under 35 U.S.C. 120, which is a non-provisionalapplication filed under 37 CFR 1.53(b)(1), claiming priority under 35USC 119(e) to provisional application No. 60/267,977 filed Feb. 9, 2001,and provisional application No. 60/287,072 filed Apr. 27, 2001, thecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a crystalline form of human insulin-likegrowth factor-1 (IGF-1) and more particularly to a crystal of humanIGF-1, a method of crystallization thereof, and its structure, obtainedby x-ray diffraction. In addition, the invention relates to methods ofidentifying new IGF-1 agonist molecules based on biophysical andbiochemical data suggesting that a single detergent molecule thatcontacts residues known to be important for IGF-1 binding protein(IGFBP) interactions binds to IGF-1 specifically, and blocks binding ofIGFBP-1 and IGFBP-3.

2. Description of Related Disclosures

There is a large body of literature on the actions and activities ofIGFs (IGF-1, IGF-2, and IGF variants). Human IGF-1 is a serum protein of70 amino acids and 7649 daltons with a pI of 8.4 (Rinderknecht andHumbel, Proc. Natl. Acad. Sci. USA, 73: 4379–4381 (1976); Rinderknechtand Humbel, J. Biol. Chem., 253: 2769 (1978)) belonging to a family ofsomatomedins with insulin-like and mitogenic biological activities thatmodulate the action of growth hormone (GH) (Van Wyk et al., Recent Prog.Horm. Res 30: 259 (1974); Binoux, Ann. Endocrinol., 41: 157 (1980);Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1981); Baxter,Adv. Clin. Chem., 25: 49 (1986); U.S. Pat. No. 4,988,675; WO 91/03253;WO 93/2307 1). IGFs share a high sequence identity with insulin, beingabout 49% identical thereto. Unlike insulin, however, which issynthesized as a precursor protein containing a 33-amino-acid segmentknown as the C-peptide (which is excised to yield a covalently linkeddimer of the remaining A and B chains), IGFs are single polypeptides(see FIG. 1).

In the developing embryo, the absence of IGF-1 leads to severe growthretardation that continues post-natally (Baker et al., Cell, 75: 73–82(1993); Powell-Braxton et al., Genes Dev., 7: 2609–2617 (1993); Liu etal., Cell, 75: 59–72 (1993); Liu et al., Molecular Endocrinol., 12:1452–1462 (1998)). While most (greater than 75%) of serum IGF-1 isproduced by the liver in response to growth hormone, this liver-derivedIGF-1 has been shown to be unnecessary for post-natal body growth inmice (Sjogren et al., Proc. Natl. Acad. Sci. USA, 96: 7088–7092 (1999)).Rather, it is the locally produced, non-hepatic IGF-1, acting in aparacrine/autocrine manner, which appears to be responsible for most ofthe post-natal growth-promoting effects of IGF-1 (Schlechter et al.,Proc. Natl. Acad. Sci. USA, 83: 7932–7934 (1986); Isaksson et al.,Science, 216: 1237–1239 (1982)). Consistent with its growth-promotingeffects, IGF-1 is a powerful mitogen, regulating diverse cellularfunctions such as cell-cycle progression, apoptosis, and cellulardifferentiation (reviewed in Jones and Clemmons, Endocr. Rev., 16: 3–34(1995) and in LeRoith, Endocrinology, 141: 1287–1288 (2000)).

IGFs have been implicated in a variety of cellular functions and diseaseprocesses, including cell cycle progression, proliferation,differentiation, and insulin-like effects in insulin-resistant diabetes.Thus, IGF has been suggested as a therapeutic tool in a variety ofdiseases and injuries. Due to this range of activities, IGF-1 has beentested in mammals for such widely disparate uses as wound healing,treatment of kidney disorders, treatment of diabetes, reversal ofwhole-body catabolic states such as ADS-related wasting, treatment ofheart conditions such as congestive heart failure, and treatment ofneurological disorders (Guler et al., Proc. Natl. Acad. Sci. USA, 85:4889–4893 (1988); Schalch et al., J. Clin. Metab., 77 1563–1568 (1993);Froesch et al., Horm. Res., 42: 66–71 (1994); Vlachopapadopoulou et al.,J. Clin. Endo. Metab., 12: 3715–3723 (1995); Saad et al., Diabetologia,37 Abstract 40 (1994); Schoenle et al., Diabetologia, 34: 675–679(1991); Morrow et al., Diabetes, 42 (Suppl.): 269 (1993) (abstract);Kuzuya et al., Diabetes, 24: 696–705 (1993); Schalch et al., “Short-termmetabolic effects of recombinant human insulin-like growth factor I(rhIGF-I) in type II diabetes mellitus”, in: Spencer E M, ed., ModernConcepts of Insulin-like Growth Factors (New York: Elsevier: 1991) pp.705–713; Zenobi et al., J. Clin. Invest., 90 2234–2241 (1992); Elahi etal., “Hemodynamic and metabolic responses to human insulin-like growthfactor-1 (IGF-I) in men,” in: Modern Concepts of Insulin-Like GrowthFactors, Spencer, E M, ed. (Elsevier: New York, 1991), pp. 219–224; Quinet al., New Engl. J. Med., 323: 1425–1426 (1990); Schalch et al.,“Short-term metabolic effects of recombinant human insulin-like growthfactor 1 (rhIGF-I) in type II diabetes mellitus,” in: Modem Concepts ofInsulin-Like Growth Factors, Spencer, E M, ed., (Elsevier: New York,1991), pp. 705–713; Schoenle et al., Diabetologia, 34: 675–679 (1991);Usala et al., N. Eng. J. Med., 327: 853–857 (1992); Lieberman et al., J.Clin. Endo. Metab., 75: 30–36 (1992); Zenobi et al., J. Clin. Invest.,90: 2234–2241 (1992); Zenobi et al., J. Clin. Invest., 89: 1908–1913(1992); Kerr et al., J. Clin. Invest., 91: 141–147 (1993); Jabri et al.,Diabetes 43: 369–374 (1994); Duerr et al., J. Clin. Invest., 95: 619–627(1995); Bondy, Ann Intern. Med., 120: 593–601 (1994); Hammerman andMiller, Am. J. Physiol., 265: F1–F14 (1993); Hammerman and Miller, J.Am. Soc. Nephrol., 5: 1–11(1994); and Barinaga et al., Science, 264:772–774 (1994)).

The patent literature also abounds with disclosures of various uses ofIGF-1, or compounds that increase active concentration of IGF-1, totreat mammals, especially human patients, for example, U.S. Pat. Nos.5,714,460; 5,273,961; 5,466,670; 5,126,324; 5,187,151; 5,202,119;5,374,620; 5,106,832; 4,988,675; 5,106,832; 5,068,224; 5,093,317;5,569,648; and 4,876,242; WO 92/11865; WO 96/01124; WO 91/03253; WO93/25219; WO 93/08826; and WO 94/16722.

The IGF system is also composed of membrane-bound receptors for IGF-1,IGF-2, and insulin. The Type 1 IGF receptor (IGF-1R) is closely relatedto the insulin receptor in structure and shares some of its signalingpathways (Jones and Clemmons, supra). The IGF-2 receptor is a clearancereceptor that appears not to transmit an intracellular signal (Jones andClemmons, supra). Since IGF-1 and IGF-2 bind to IGF-1R with a muchhigher affinity than to the insulin receptor (Cascieri et al.,Biochemistry, 27: 3229–3233 (1988)), it is most likely that most of theeffects of IGF-1 and IGF-2 are mediated by IGF-1R (Humbel, Eur. J.Biochem. 190:445–462 (1990); Ballard et al., “Does IGF-1 ever actthrough the insulin receptor?”, in Baxter et al. (Eds.), TheInsulin-Like Growth Factors and Their Regulatory Proteins, (Amsterdam:Elsevier, 1994), pp. 131–138).

IGF-1R is an α2β2 heterotetramer of disulfide-linked α and β subunits.αβ dimers are themselves disulfide linked on the cell surface to form acovalent heterotetramer. As in the insulin/insulin receptor complex,IGF-1 binds to the IGF-1R with a 1:2 stoichiometry (De Meyts,Diabetologia, 37: S135-S148 (1994)), with a high affinity site (K_(d)about 0.4 nM) and a low affinity site (K_(d) about 6 nM) (Tollefsen andThompson, J. Biol. Chem., 263: 16267–16273 (1988)). The x-ray crystalstructure of the first three domains of IGF-1R has been determined(Garrett et al., Nature, 394, 395–399 (1998)). It contains threedistinct domains (L1, Cys-rich, L2). Mutations that affect IGF-1 bindingmap to the concave surface of the receptor.

IGF-1R is a key factor in normal cell growth and development (Isakssonet al., Endocrine Reviews, 8: 426–438 (1987); Daughaday and Rotwein,Endocrine Rev., 10:68–91 (1989)). Increasing evidence suggests, however,that IGF-1R signaling also plays a critical role in growth of tumorcells, cell transformation, and tumorigenesis (Baserga, Cancer Res.,55:249–252 (1995)). Key examples include loss of metastatic phenotype ofmurine carcinoma cells by treatment with antisense RNA to the IGF-1R(Long et al., Cancer Res., 55:1006–1009 (1995)) and the in vitroinhibition of human melanoma cell motility (Stracke et al., J Biol.Chem., 264:21544–21549 (1989)) and of human breast cancer cell growth bythe addition of IGF-1R antibodies (Rohlik et al., Biochem. Biophys. Res.Commun., 149:276–281 (1987)).

The IGFs are potent breast cancer cell mitogens based on the observationthat IGF-1 enhanced breast cancer cell proliferation in vitro (Cullen etal., Cancer Res., 50:48–53 (1990)). Breast cancers express IGF-2 andIGF-1R, providing all the required effectors for an autocrine-loop-basedproliferation paradigm (Quinn et al., J. Biol. Chem., 271:11477–11483(1996); Steller et al., Cancer Res., 56:1761–1765 (1996)). Becausebreast cancer is a common malignancy affecting approximately one inevery eight women and is a leading cause of death from cancer in NorthAmerican women (LeRoith et al., Ann. Int. Med., 122:54–59 (1995)), newrational therapies are required for intervention. IGF-1 can suppressapoptosis, and therefore cells lacking IGF-1Rs or having compromisedIGF-1R signaling pathways may give rise to tumor cells that selectivelydie via apoptosis (Long et al., Cancer Res., 55:1006–1009 (1995)).Furthermore, it has recently become evident that alterations in IGFsignaling in the context of other disease states, such as diabetes, maybe responsible for exacerbating the complications of retinopathy (Smithet al., Science, 276:1706–1709 (1997)) and nephropathy (Horney et al.,Am. J. Physiol. 274: F1045–F1053 (1998)).

IGF-1 in vivo is mostly found in complex with a family of at least sixserum proteins known as IGFBPs (Jones and Clemmons, supra; Bach andRechler, Diabetes Reviews, 3: 38–61 (1995)), that modulate access of theIGFs to the IGF-1R. They also regulate the concentrations of IGF-1 andIGF-2 in the circulation and at the level of the tissue IGF-1 R(Clemmons et al., Anal. NY Acad. Sci. USA, 692:10–21 (1993)). The IGFBPsbind IGF-1 and IGF-2 with varying affinities and specificities (Jonesand Clemmons, supra; Bach and Rechler, supra). For example, IGFBP-3binds IGF-1 and IGF-2 with a similar affinity, whereas IGFBP-2 andIGFBP-6 bind IGF-2 with a much higher affinity than they bind IGF-1(Bach and Rechler, supra; Oh et al., Endocrinology, 132, 1337–1344(1993)): The major carrier protein is IGFBP-3. Nothing is currentlyknown about the stoichiometry of binding in these complexes of IGF-1 andits IGFBPs, due to the heterogeneous size of the complexes caused byglycosylation.

IGF-1 naturally occurs in human body fluids, for example, blood andhuman cerebral spinal fluid. Although IGF-1 is produced in many tissues,most circulating IGF-1 is believed to be synthesized in the liver. TheIGFBPs are believed to modulate the biological activity of IGF-1 (Jonesand Clemmons, supra), with IGFBP-1 (Lee et al., Proc. Soc. Exp. Biol. &Med., 204: 4–29 (1993)) being implicated as the primary binding proteininvolved in glucose metabolism (Baxter, “Physiological roles of IGFbinding proteins”, in: Spencer (Ed.), Modern Concepts of Insulin-likeGrowth Factors (Elsevier, New York, 1991), pp. 371–380). IGFBP-1production by the liver is regulated by nutritional status, with insulindirectly suppressing its production (Suikkari et al., J. Clin.Endocrinol. Metab., 66: 266–272 (1988)).

The function of IGFBP-1 in vivo is poorly understood. The administrationof purified human IGFBP-1 to rats has been shown to cause an acute, butsmall, increase in blood glucose (Lewitt et al, Endocrinology, 129:2254–2256 (1991)). The regulation of IGFBP-1 is somewhat betterunderstood. It has been proposed (Lewitt and Baxter, Mol. CellEndocrinology, 79: 147–152 (1991)) that when blood glucose rises andinsulin is secreted, IGFBP-1 is suppressed, allowing a slow increase in“free” IGF-1 levels that might assist insulin action on glucosetransport. Such a scenario places the function of IGFBP-1 as a directregulator of blood glucose.

In most cases, addition of exogenous IGFBP blunts the effects of IGF-1.For example, the growth- stimulating effect of estradiol on the MCF-7human breast cancer cells is associated with decreased IGFBP-3 mRNA andprotein accumulation, while the anti-estrogen ICI 182780 causes growthinhibition and increased IGFBP-3 mRNA and protein levels (Huynh et al.,J Biol. Chem., 271: 1016–1021 (1996); Oh et al., Prog. Growth FactorRes., 6:503–512 (1995)). It has also been reported that the in vitroinhibition of breast cancer cell proliferation by retinoic acid mayinvolve altered IGFBP secretion by tumor cells or decreased circulatingIGF-1 levels in vivo (LeRoith et al., Ann. mt. Med., 122:54–59 (1995);Oh et al., (1995), supra). Contrary to this finding, treatment of MCF-7cells with the anti-estrogen tamoxifen decreases IGF-1R signaling in amanner that is unrelated to decreased IGFBP production (Lee et al., JEndocrinol., 152:39 (1997)). Additional support for the generalanti-proliferative effects of the IGFBPs is the striking finding thatIGFBP-3 is a target gene of the tumor suppressor, p53 (Buckbinder etal., Nature, 377:646–649 (1995)). This suggests that the suppressoractivity of p53 is, in part, mediated by IGFBP-3 production and theconsequential blockade of IGF action (Buckbinder et al., supra). Theseresults indicate that the IGFBPs can block cell proliferation bymodulating paracrine/autocrine processes regulated by IGF-1/IGF-2. Acorollary to these observations is the finding that prostate-specificantigen (PSA) is an IGFBP-3-protease, which upon activation, increasesthe sensitivity of tumor cells to the actions of IGF-1 /IGF-2 due to theproteolytic inactivation of IGFBP-3 (Cohen et al., J. Endocr.,142:407–415 (1994)). The IGFBPs complex with IGF-1/IGF-2 and interferewith the access of IGF-1/IGF-2 to IGF-1Rs (Clemmons et al., Anal. NYAcad. Sci. USA, 692:10–21 (1993)). IGFBP-1, -2 and -3 inhibit cellgrowth following addition to cells in vitro (Lee et al., J Endocrinol.,152:39 (1997); Feyen et al., J Biol. Chem., 266:19469–19474 (1991)).Further, IGFBP-1 (McGuire et al., J Natl. Cancer Inst.,84:1336–1341(1992); Figueroa et al., J Cell Physiol., 157:229–236(1993)), IGFBP-3 (Oh et al. (1995), supra; Pratt and Pollak, Biophys.Res. Commun., 198:292–297 (1994)) and IGFBP-2 have all been shown toinhibit IGF-1 or estrogen-induced breast cancer cell proliferation atnanomolar concentrations in vitro. These findings support the idea thatthe IGFBPs are potent antagonists of IGF action. There is also evidencefor a direct effect of IGFBP-3 on cells through its own cell surfacereceptor, independent of IGF interactions (Oh et al., J Biol. Chem.,268:14964–14971 (1993); Valentinis et al., Mol. Endocrinol., 9:361–367(1995)). Taken together, these findings underscore the importance of IGFand IGF-1R as targets for therapeutic use.

IGFs have mitogenic and anti-apoptotic influences on normal andtransformed prostate epithelial cells (Hsing et al., Cancer Research,56: 5146(1996); Culig et al., Cancer Research, 54: 5474(1994); Cohen etal., Hormone and Metabolic Research, 26:81 (1994); Iwamura et al.,Prostate, 22:243 (1993); Cohen et al., J. Clin. Endocrin. & Metabol.,73:401 (1991); Rajah et al., J. Biol. Chem., 272: 12181 (1997)). Mostcirculating IGF-1 originates in the liver, but IGF bioactivity intissues is related not only to levels of circulating IGFs and IGFBPs,but also to local production of IGFs, IGFBPs, and IGFBP proteases (Jonesand Clemmons, supra). Person-to-person variability in levels ofcirculating IGF-1 and IGFBP-3 (the major circulating IGFBP (Jones andClemmons, supra)) is considerable (Juul et al., J. Clin. Endocrinol. &Metabol., 78: 744 (1994); Juul et al., J. Clin. Endocrinol. & Metabol.,80: 2534 (1995)), and heterogeneity in serum IGF-1 levels appears toreflect heterogeneity in tissue IGF bioactivity. Markers relating toIGF-axis components can be used as a risk marker for prostate cancer, asPSA is likewise used (WO 99/38011).

Unlike most other growth factors, the IGFs are present in highconcentrations in the circulation, but only a small fraction of the IGFsis not protein bound. For example, it is generally known that in humansor rodents, less than 1% of the IGFs in blood is in a “free” or unboundform (Juul et al., Clin. Endocrinol., 44: 515–523 (1996); Hizuka et al.,Growth Regulation, 1: 51–55 (1991); Hasegawa et al., J. Clin.Endocrinol. Metab., 80: 3284–3286 (1995)). The overwhelming majority ofthe IGFs in blood circulate as part of a non-covalently associatedternary complex composed of IGF-1 or IGF-2, IGFBP-3, and a large proteintermed the acid-labile subunit (ALS). The ternary complex of an IGF,IGFBP-3, and ALS has a molecular weight of approximately 150,000daltons, and it has been suggested that the function of this complex inthe circulation may be to serve as a reservoir and buffer for IGF-1 andIGF-2, preventing rapid changes in free IGF-1 or IGF-2.

There has been much work identifying the regions on IGF-1 and IGF-2 thatbind to the IGFBPs (Bayne et al, J. Biol. Chem., 265: 15648–15652(1990); Dubaquie and Lowman, Biochemistry, 38: 6386–6396 (1999); andU.S. Pat. Nos. 5,077,276; 5,164,370; and 5,470,828). For example, it hasbeen discovered that the N-terminal region of IGF-1 and IGF-2 iscritical for binding to the IGFBPs (U.S. Pat. Nos. 5,077,276; 5,164,370;and 5,470,828). Thus, the natural IGF-1 variant, designated des (1-3)IGF-1, binds poorly to IGFBPs.

A similar amount of research has been devoted to identifying the regionson IGF-1 and IGF-2 that bind to IGF-1R (Bayne et al., supra; Oh et al.,Endocrinology (1993), supra). It was found that the tyrosine residues inIGF-1 at positions 24, 31, and 60 are crucial to the binding of IGF-1 toIGF-1R (Bayne et al., supra). Mutant IGF-1 molecules where one or moreof these tyrosine residues are substituted showed progressively reducedbinding to IGF-1R. Bayne et al., supra, also investigated whether suchmutants of IGF-1 could bind to IGF-1R and to the IGFBPs. They found thatquite different residues on IGF-1 and IGF-2 are used to bind to theIGFBPs from those used to bind to IGF-1R. It is therefore possible toproduce IGF variants that show reduced binding to the IGFBPs, but,because they bind well to IGF-1R, show maintained activity in in vitroactivity assays.

Also reported was an IGF variant that binds to IGFBPs but not to IGFreceptors and therefore shows reduced activity in in vitro activityassays (Bar et al., Endocrinology, 127: 3243–3245 (1990)). In thisvariant, designated (1–27, gly⁴, 38–70)-hIGF-1, residues 28–37 of theC-region of human IGF-l (SEQ ID NO: 1) are replaced by a four-residueglycine bridge.

Other truncated IGF-1 variants are disclosed. For example, in the patentliterature, WO 96/33216 describes a truncated variant having residues1–69 of authentic IGF-1 (SEQ ID NO: 1). EP 742,228 discloses two-chainIGF-1 superagonists, which are derivatives of the naturally occurring,single-chain IGF-1 having an abbreviated C-region. The IGF-1 analogs areof the formula: BC^(n), A wherein B is the B-region of IGF-1 or afunctional analog thereof, C is the C- region of IGF-1 (SEQ ID NO: 1) ora functional analog thereof, n is the number of amino acids in theC-region and is from about 6 to about 12, and A is the A-region of IGF-1or a functional analog thereof.

Additionally, Cascieri et al., Biochemistry, 27: 3229–3233 (1988)discloses four mutants of IGF-1 (SEQ ID NO: 1), three of which havereduced affinity to IGF-1R. These mutants are: (Phe²³, Phe²⁴,Tyr²⁵)IGF-1 (which is equipotent to human IGF-1 in its affinity to theTypes 1 and 2 IGE and insulin receptors), (Leu²⁴)IGF-1 and (Ser²⁴)IGF-1(which have a lower affinity than IGF-1 to the human placental IGF-1R,the placental insulin receptor, and the IGF-1R of rat and mouse cells),and desoctapeptide (Leu²⁴)IGF-1 (in which the loss of aromaticity atposition 24 is combined with the deletion of the carboxyl-terminalD-region of hIGF-1 (SEQ ID NO: 1), which has lower affinity than(Leu24)IGF-1 for the IGF-1R and higher affinity for the insulinreceptor). These four mutants have normal affinities for human serumbinding proteins.

Bayne et al., J. Biol. Chem., 263: 6233–6239 (1988) discloses fourstructural analogs of human IGF-1 (SEQ ID NO: 1): a B-chain mutant inwhich the first 16 amino acids of IGF-1 were replaced with the first 17amino acids of the B-chain of insulin, (Gln³, Ala⁴)IGF-1, (Tyr¹⁵,Leu¹⁶)IGF-1, and (Gln³, Ala⁴, Tyr⁵, Leu¹⁶)IGF-1. These studies identifysome of the regions of IGF-1 that are responsible for maintaininghigh-affinity binding with the serum binding protein and the Type 2 IGFreceptor.

In another study, Bayne et al., J. Biol. Chem., 264: 11004–11008 (1988)discloses three structural analogs of IGF-1 (SEQ ID) NO: 1):(1–62)IGF-1, which lacks the carboxyl-terminal 8-amino-acid D-region ofIGF-1; (1–27, Gly⁴, 38–70)IGF-1, in which residues 28–37 of the C-regionof IGF-1 are replaced by a four-residue glycine bridge; and (1–27, Gly⁴,38–62)IGF-1, with a C-region glycine replacement and a D-regiondeletion. Peterkofsky et al., Endocrinology, 128: 1769–1779 (1991)discloses data using the Gly⁴ mutant of Bayne et al., supra (vol. 264).

Cascieri et al., J. Biol. Chem., 264: 2199–2202 (1989) discloses threeIGF-1 analogs in which specific residues in the A-region of IGF-1 (SEQID NO: 1) are replaced with the corresponding residues in the A chain ofinsulin. The analogs are: (Ile⁴¹, Glu⁴⁵, Gln⁴⁶, Thr⁴⁹, Ser⁵⁰, Ile⁵¹,Ser⁵³, Tyr⁵⁵, Gln⁵⁶)IGF-1, an A-chain mutant in which residue 41 ischanged from threonine to isoleucine and residues 42–56 of the A-regionare replaced; (Thr⁴⁹, Ser⁵⁰,Ile⁵¹)IGF-1; and (Tyr⁵⁵, Gln⁵⁶)IGF-1.

Clemmons et al., J. Biol. Chem., 265: 12210–12216 (1990) discloses useof IGF-1 analogs that have reduced binding affinity for either IGF-1R orbinding proteins to study the ligand specificity of IGFBP-1 and the roleof IGFBP-1 in modulating the biological activity of IGF-1.

WO 94/04569 discloses a specific binding molecule, other than a naturalIGFBP, that is capable of binding to IGF-1 and can enhance thebiological activity of IGF-1.

Peptides that bind to IGFBP-1, block IGF-1 binding to this bindingprotein, and thereby release “free-IGF” activity from mixtures of IGF-1and IGFBP-1 have been recently described (Lowman et al., Biochemistry,37: 8870–8878 (1998); WO 98/45427 published Oct. 15, 1998; Lowman etal., International Pediatric Nephrology Association, Fifth Symposium onGrowth and Development in Children with Chronic Renal Failure (New York,Mar. 13, 1999)). Also described is the natural molecule, des(1-3)IGF-1,which shows selectively reduced affinity for some of the IGF bindingproteins, yet a maintained affinity for the IGF receptor (U.S. Pat. Nos.5,077,276; 5,164,370; 5,470,828).

Exploitation of the interaction between IGF and IGFBP in screening,preventing, or treating disease has been limited, however, because of alack of specific antagonists. To date, only one publication is known toexist that describes the application of an IGF-1/IGF-2 antagonist as apotential therapeutic adjunct in the treatment of cancer (Pietrzkowskiet al., Cancer Res., 52: 6447-6451 (1992)). In that report, a peptidecorresponding to the D-region of IGF-1 was synthesized for use as anIGF-1/2 antagonist. This peptide exhibited questionable inhibitoryactivity against IGF-1. The basis for the observed inhibition isunclear, as the D-region does not play a significant role in IGF-1Rbinding but rather, in IGF-1 binding to the insulin receptor (Cooke etal., Biochem., 30:5484–5491 (1991); Bayne et al., supra (Vol. 264); Yeeet al., Cell Growth and Different., 5:73–77 (1994)).

WO 00/23469 discloses the portions of IGFBP and IGF peptides thataccount for IGF-IGFBP binding, i.e., an isolated IGF binding domain ofan IGFBP or modification thereof that binds IGF with at least about thesame binding affinity as the full-length IGFBP. The patent publicationalso discloses an IGF antagonist that reduces binding of IGF to an IGFreceptor, and/or binds to a binding domain of IGFBP.

Additionally, WO 93/23067 discloses pharmaceutical compositionscomprising short peptides that function as IGF-1 receptor antagonists.The peptides used in the pharmaceutical compositions consist of lessthan 25 amino acids, comprise at least a portion of the C- or D-regionfrom IGF-1, and inhibit IGF-1-induced autophosphorylation of IGE-1receptors.

Polypeptides, including the IGF molecules, have a three-dimensionalstructure determined by the primary amino acid sequence and theenvironment surrounding the polypeptide. This three-dimensionalstructure establishes the activity, stability, binding affinity, bindingspecificity, and other biochemical attributes of the polypeptide. Thus,knowledge of the three-dimensional structure of a protein can providemuch guidance in designing agents that mimic, inhibit, or improve itsbiological activity in soluble or membrane-bound forms.

The three-dimensional structure of a polypeptide may be determined in anumber of ways. Many of the most precise methods employ x-raycrystallography (Van Holde, Physical Biochemistry (Prentice Hall: N.J.,1971), pp. 221–239). This technique relies on the ability of crystallinelattices to diffract x-ray or other forms of radiation. Diffractionexperiments suitable for determining the three-dimensional structure ofmacromolecules typically require high-quality crystals. Unfortunately,such crystals have been unavailable for IGF-1 as well as many otherproteins of interest. Crystals have been described for M-CSF (EP668,914B1), CD40 ligand (WO 97/00895), and a BC2 Fab fragment (WO99/01476), for example.

The crystallization of insulin is an intensively researched field, bothwith respect to work on structural analysis (Adams et al., Nature,224:491 (1969)) and pharmaceutical applications. Examples of insulincrystal suspensions that are used therapeutically include suspensions ofrhombohedral zinc-insulin crystals that are stable in the presence of0.8 to 2.5% of zinc (based on the weight of insulin) at a neutral pHvalue and exhibit a delayed action, and isophane insulin protaminecrystals, which are used in delayed action products in the form of smallrods. A few other crystal modifications of insulin are furthermoreknown, but these have hitherto been of interest only for X-ray structureanalysis. Thus, zinc-free orthorhombic and monoclinic crystals have beenobtained under acid pH conditions (Einstein and Low, Acta Crystallogr.,15: 32–34 (1962)). Smaller rhombic dodecahedra, which are to beclassified in the cubic space group, have been obtained at theisoelectric point, also in the absence of zinc. Finally, a monocliniccrystal form of insulin has been obtained above the isoelectric point inthe presence of zinc and in the presence of phenol or phenolderivatives. These crystals grow to a considerable size (up to 3 mm)within a few days and have sharp edges. Interestingly, these crystalshave been found only on glass surfaces and not on the free surface ofthe solution. Crystal suspensions and other crystal forms of insulinpreparations and insulin analogs are described, for example, in suchrepresentative patents as U.S. Pat. Nos. 4,959,351; 5,840,680;5,834,422; 6,127,334; 5,952,297; 5,650,486; 5,898,028; 5,898,067;5,948,751; 5,747,642; 5,597,893; 5,547,930; 5,534,488; 5,504,188;5,461,031; and 5,028,587.

Various methods for preparing crystalline proteins and polypeptides areknown in the an (McPherson et al., “Preparation and Analysis of ProteinCrystals,” McPherson (Robert E. Krieger Publishing Company, Malabar,Fla., 1989); Weber, Advances in Protein Chemistry, 41: 1–36 (1991); U.S.Pat. Nos. 4,672,108 and 4,833,233). Although there are multipleapproaches to crystallizing polypeptides, no single set of conditionsprovides a reasonable expectation of success, especially when thecrystals must be suitable for x-ray diffraction studies. Significanteffort is required to obtain crystals of sufficient size and resolutionto provide accurate information regarding the structure. For example,once a protein of sufficient purity is obtained, it must be crystallizedto a size and clarity that is useful for x-ray diffraction andsubsequent structure resolution. Further, although the amino acidsequence of a target protein may be known, this sequence informationdoes not allow an accurate prediction of the crystal structure of theprotein. Nor does the sequence information afford an understanding ofthe structural, conformational, and chemical interactions between aligand such as an IGFBP and its protein target. Thus, although crystalstructures can provide a wealth of valuable information in the field ofdrug design and discovery, crystals of certain biologically relevantcompounds such as IGF-1 are not readily available to those skilled inthe art. High-quality, diffracting crystals of IGF-1 would assist thedetermination of its three-dimensional structure.

Generation of specific IGF-1 antagonists has been restricted, at leastin part, because of difficulties in studying the structure of IGF andIGFBPs. Due to the inability to obtain crystals of IGF-1 suitable fordiffraction studies, for example, an extrapolation of IGF-1 structurebased on the crystal structure of porcine insulin was the most importantstructural road map for IGF-1 available (Blundell et al., Proc. Natl.Acad. Sci. USA, 75:180–184 (1978)). See also Blundell et al., Fed.Proc., 42: 2592–2597 (1983), which discloses tertiary structures,receptor binding, and antigenicity of IGFs. Based on studies ofchemically modified and mutated IGF-1, a number of common residuesbetween IGF-1 and insulin have been identified as being part of theIGF-1R-insulin receptor contact site, in particular, the aromaticresidues at positions 23–25.

Using NMR and restrained molecular dynamics, the solution structure ofIGF-1 was recently reported (Cooke et al., supra). The resultingminimized structure was shown to better fit the experimental findings onmodified IGF-1, as well as the extrapolations made from thestructure-activity studies of insulin. Further, De Wolf et al, ProteinSci., 5: 2193–2202 (1996) discloses the solution structure of amini-IGF-1. Sato et al., Int. J. Pept. Protein Res., 41: 433–440 (1993)discloses the three-dimensional structure of IGF-1 determined by 1H-NMRand distance geometry. Laajoki et al., J. Biol. Chem., 275: 10009–10015(2000) discloses the solution structure and backbone dynamics oflong-[Arg(3)]IGF-1. See also Laajoki et al., FEBS Lett., 420: 97–102(1997)). The small number of NMR models available for IGF-1 are not verywell defined, as there are large RMSDs between the backbone atoms of thehelical segments. The best NMR model is of IGF-2 in which threealpha-helices are shown. See Torres et al., J. Mol. Biol., 248: 385–401(1995), which discloses the solution structure of human IGF-2 and itsrelationship to receptor and binding protein interactions. In allstructures, the C- and D-regions are very poorly defined.

In addition to providing structural information, crystallinepolypeptides provide other advantages. For example, the crystallizationprocess itself further purifies the polypeptide and satisfies one of theclassical criteria for homogeneity. In fact, crystallization frequentlyprovides unparalleled purification quality, removing impurities that arenot removed by other purification methods such as HPLC, dialysis,conventional column chromatography, etc. Moreover, crystallinepolypeptides are often stable at ambient temperatures and free ofprotease contamination and other degradation associated with solutionstorage. Crystalline polypeptides may also be useful as pharmaceuticalpreparations. Finally, crystallization techniques in general are largelyfree of problems such as denaturation associated with otherstabilization methods (e.g. lyophilization). Thus, there exists asignificant need for preparing IGF-1 compositions in crystalline formand determining their three-dimensional structure. The present inventionfulfills this and other needs. Once crystallization has beenaccomplished, crystallographic data provides useful structuralinformation that may assist the design of peptides that may serve asagonists or antagonists. In addition, the crystal structure providesinformation useful to map the receptor-binding domain, which could thenbe mimicked by a small non-peptide molecule that may serve as anantagonist or agonist. Also, findings regarding the detergent'sinhibition of the binding of IGFBP to IGF-1 can be used to identify newIGF-1 agonists.

SUMMARY OF THE INVENTION

Accordingly, the invention is as claimed. IGF-1 has been crystallizedand its structure determined using multiwavelength anomalous diffraction(MAD) at 1.8 angstroms resolution by exploiting the anomalous scatteringof a single bromide ion and six of the seven sulfur atoms of IGF-1. TheC-region of IGF-1, which is ordered in the crystal structure, forms atype II beta-turn and mediates a crystal packing interaction across acrystallographic dyad. The solution state of IGF-1 was characterized byanalytical ultracentrifugation, and the results indicate that IGF-1exists primarily as a monomer at neutral pH, with only a slight tendencyto dimerize at millimolar concentrations. A molecule of detergent,N,N-bis(3-D-gluconamidopropyl)-deoxycholamine (deoxy big CHAPS),mediates a crystal packing contact between symmetry-related molecules.Biophysical and biochemical data show that theN,N-bis(3-D-gluconamidopropyl)-deoxycholamine binds to IGF-1specifically and blocks binding of IGFBP-1 and IGFBP-3.

Accordingly, in one aspect, the invention provides a crystal formed byIGF-1 that diffracts x-ray radiation to produce a diffraction patternrepresenting the three-dimensional structure of the IGF-1. Preferablythis crystal has approximately the following cell constants a=31.831 Å,b=71.055 Å, c=65.995 Å, and a space group of C222₁. Also preferably, theIGF-1 contains an A-, B-, C-, and D-region and forms a dimer in thecrystal, and further preferred is the crystal comprising a receptorbinding site at the dimer interface.

The invention also provides a composition comprising the above crystal.Preferably in this composition the IGF-1 is biologically active whenresolubilized. The invention further provides a method of treating amammal suffering from an agonist disorder, preferably a human patient,said method comprising administering to said mammal an effective amountof the above resolubilized composition.

The invention also provides a method of crystallizing IGF-1 comprisingthe steps of:

(a) mixing an aqueous solution comprising IGF-1 with a reservoirsolution comprising a precipitant to form a mixed volume; and

(b) crystallizing the mixed volume.

The invention also provides crystalline IGF-1 produced by the abovemethod.

Additionally, the invention provides a method for determining athree-dimensional structure of IGF-1 comprising:

-   -   (a) crystallizing the IGF-1;    -   (b) irradiating the crystalline IGF-1 to obtain a diffraction        pattern characteristic of the crystalline IGF-1; and

(c) transforming the diffraction pattern into the three-dimensionalstructure of the IGF-1. Further, the invention provides amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data that, when read by an appropriatemachine, displays a three-dimensional representation of a crystal of amolecule comprising IGF-1.

In further aspects, the invention provides an IGF-1 crystal with thestructural coordinates shown in Appendix 1.

Additionally, the invention provides a method of using athree-dimensional structure of IGF-1 derived from an IGF-1 crystalwherein the three-dimensional structure of IGF-1 includes an IGF-1receptor-binding region, the method comprising identifying compoundshaving structures that interact with the receptor-binding region of thethree-dimensional structure of IGF-1 and function as an IGF-1 agonist orantagonist. Preferably in such method the three-dimensional structure ofIGF-1 includes alpha-carbon coordinates substantially the same as thoseof the structural information presented in Appendix 1.

In another aspect, the invention provides a method of identifying IGF-1agonists or antagonists comprising the steps of:

-   -   (a) crystallizing IGF-1 to form IGF-1 crystals, the IGF-1        crystals containing a group of amino acid residues defining an        IGF-1 receptor-binding region;    -   (b) irradiating the IGF-1 crystals from step (a) to obtain a        diffraction pattern of the IGF-1 crystals;    -   (c) determining a three-dimensional structure of IGF-1 from the        diffraction pattern, the structure including an IGF-1        receptor-binding region; and    -   (d) identifying an IGF-1 agonist or antagonist having a        three-dimensional structure that functionally duplicates        essential IGF receptor-binding, solvent-accessible residues        presenting the three-dimensional structure of the IGF-1        receptor-binding region, said IGF-1 agonist or antagonist having        altered signal transduction capacity to IGF-1-responsive cells,        as compared to IGF-1.

Preferably, in this method the solvent-accessible residues do notparticipate in formation of the IGF-1 interface.

According to certain further aspects, the invention includes a method ofdesigning a compound, such as a peptidomimetic, that mimics the3-dimensional surface structure of IGF-1 comprising the steps of:

(a) determining the 3-dimensional structure of the IGF-1; and

(b) designing a compound that mimics the 3-dimensional surface structureof the IGF-1.

According to a further embodiment, the invention provides a method foridentifying a peptidomimetic that binds IGF-1 and blocks binding of anIGFBP or a receptor that binds to IGF-1 comprising the steps of:

(a) searching a molecular structure database with the structuralparameters or structural coordinates provided in Appendix 1; and

(b) selecting a molecule from the database that mimics the structuralparameters or structural coordinates of the IGF-1.

The invention also provides a method for determining at least a portionof a three-dimensional structure of a molecular complex, said complexcomprising IGF-1 and said method comprising the steps of:

-   -   (a) determining the structural coordinates of a crystal of        IGF-1;    -   (b) calculating phases from the structural coordinates;    -   (c) calculating an electron density map from the phases obtained        in step (b); and    -   (d) determining the structure of at least a portion of the        complex based on said electron density map.

Preferably the structural coordinates used in step (a) are substantiallythe same as those described in Appendix 1 or describe substantially thesame crystal as the coordinates in Appendix 1.

The invention also provides a method for evaluating the ability of achemical entity to associate with IGF-1 or a complex thereof, the methodcomprising the steps of:

-   -   (a) employing computational or experimental means to perform a        fitting operation between the chemical entity and the IGF-1 or        complex thereof, thereby obtaining data related to the        association; and    -   (b) analyzing the data obtained in step (a) to determine the        characteristics of the association between the chemical entity        and the IGF-1 or complex thereof.

The invention also provides a chemical entity identified by the abovemethod that interferes with the in vivo or in vitro association betweenIGF-1 and its receptor or between IGF-1 and at least one of its bindingproteins, or associates with a binding site on IGF-1.

Also provided is a heavy-atom derivative of a crystallized form ofIGF-1.

The invention also comprises a method of computationally orexperimentally evaluating a chemical entity to obtain information aboutits association with one or more binding sites of IGF-1 using a crystalof IGF-1 having the structural coordinates described in Appendix 1.

Any peptide analogs and other chemical entities identified using theabove methods of the present invention are useful in the therapeuticmethods described herein and as pharmaceutical compositions.

The invention also provides a method of identifying indirect agonists ofIGF-1 comprising the steps of:

-   -   (a) comparing the ability of        N,N-bis(3-D-gluconamidopropyl)-deoxycholamine to inhibit binding        of IGFBP-1 or IGFBP-3 to IGF-1 with the ability of a candidate        indirect agonist of IGF-1 to so inhibit binding; and    -   (b) determining whether the candidate agonist inhibits such        binding at least as well as        N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.

In a preferred embodiment, the comparison is accomplished by competitionassay between N,N-bis(3-D-gluconamidopropyl)-deoxycholamine and thecandidate agonist. In a more preferred embodiment, inhibition of bindingis measured by pre-incubatingN,N-bis(3-D-gluconamidopropyl)-deoxycholamine or the candidate agonistwith IGF-1 expressed on bacteriophage particles and measuring residualbinding of IGF-1 to IGFBP-1 or IGFBP-3 in a plate-based ELISA assay.

The invention further provides a method of identifying indirect agonistsof IGF-1 comprising co-crystallizing a candidate indirect agonist ofIGF-1 with IGF-1 to form a co crystalline structure and determining ifthe candidate agonist binds to one or both of two patches on IGF-1 (SEQID NO: 1), wherein one patch has the amino acid residues Glu 3, Thr 4,Leu 5, Asp 12, Ala 13, Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53,Leu 54, and Leu 57, second patch has the amino acid residues Val 11, Gin15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55, and whereinbinding occurs if there is at least one contact between each listedamino acid residue of a given patch and the candidate agonist that isless than or equal to 6 angstroms in the co-crystalline structure. Inpreferred embodiments, the candidate agonist inhibits binding of IGFBP-1or -3 to IGF-1 (SEQ ID NO: 1) at least as well as N,N-bis(3-D-gluconamidopropyl)-deoxycholamine. More preferred is themethod wherein inhibition of binding is measured using a competitionassay between N, N-bis(3-D-gluconamidopropyl)- deoxycholamine and thecandidate agonist. Most preferred is the method wherein inhibition ofbinding is measured by pre-incubating N,N-bis(3-D-gluconamidopropyl)-deoxycholamine or the candidate agonistwith IGF-1 expressed on bacteriophage particles and measuring residualbinding of IGF-1 to IGFBP-1 or IGFBP-3 in a plate-based ELISA assay.

Also provided herein is a method for treating an IGF-1 agonist disorderin a mammal comprising administering to the mammal an effective amountof N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.

Further provided herein is a co-crystalline complex of IGF-1 andN,N-bis(3-D-gluconamidopropyl)-deoxycholamine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 aligns the sequences of IGF-1 (SEQ ID NO:1), IGF-2 (SEQ ID NO:2),and insulin (SEQ ID NO:3). The A-, B-, and C-chains of insulin (and thesequences of IGF-1 and IGF-2 corresponding thereto) are shownrespectively in bold, underlined, and italicized text. The threearomatic residues are shown by outlining the text. The residues markedwith a (!) have been demonstrated to be important for binding to theIGF-1 receptor. The residues marked with a “*” have been shown to beimportant for binding to IGFBP-1 and IGFBP-3. The carboxyl-terminalresidues comprising the D-region of IGF-1 and IGF-2 are depicted inregular type.

FIG. 2 is a ribbon diagram of IGF-1 showing the backbone fold. In theRamachandran plot, 97.7% is most favored and 2.3% is allowed.

FIG. 3 is a ribbon diagram of both IGF-1 (left structure) and insulin(right structure).

FIG. 4 is a ribbon diagram of IGF-1 showing that the detergent used inthe reservoir solution, (N,N-bis(3-D-gluconamidopropyl)-deoxycholamine),binds into a small hydrophilic cleft at the base of the B-helix. Thedetergent is represented by lighter gray structures than the IGF-1structures.

FIG. 5 is a ribbon diagram of IGF-1 as a dimer, with the detergent shownin lighter gray.

FIG. 6 is a ribbon diagram of IGF-1 as a dimer, showing that theresidues important for receptor binding (indicated by ring structures inthe center portion of the figure) cluster at the dimer interface. Thedetergent is shown in lighter gray at the outer portions of the figure.

FIGS. 7A and 7B are ribbon diagrams of IGF-1 (SEQ ID NO: 1)demonstrating, as does FIG. 4, that the detergent used in the reservoirsolution (N,N-bis(3-D-gluconamidopropyl)-deoxycholamine), shown in stickform, binds into a small hydrophilic cleft at the base of the B-helix.In FIG. 7A the detergent head group is inserted into the cleft lined byresidues Leu 5, Phe 16, Val 17, Leu 54, and Leu 57 of SEQ ID NO: 1. Thevarious shades of gray are according to the alanine-scanning mutagenesisresults of Dubaquie and Lowman, supra, with the Phe 16, Val 17, and Leu5 regions indicating a 5–10 fold reduction, the Glu 3 region a 10–100fold reduction, and the Pro 63 and Pro 63′ regions a >100 fold reductionin affinity for IGFBP-1 where the amino acid residues are numbered withreference to the amino acid sequence of IGF-1 (SEQ ID NO:1). The blackpart at the far right corresponds to the symmetry-related IGF-1 moleculethat forms the crystallographic dimer. The circle near Leu 54 indicatesthe C10 atom of the detergent, which differs from another detergent(3-((3-cholamidopropyl) dimethylammonio)-1-propane sulphonate; or CHAPS)by having a hydroxyl group at this position. FIG. 7B shows the view fromthe opposite surface of the detergent and depicts the interactions ofthe detergent molecule with a symmetry-related IGF-1 molecule. As inFIG. 7A the various shades of gray are according to the alanine-scanningmutagenesis results of Dubaquie and Lowman, supra, with the group nearGln 15 indicating a 5–10 fold reduction, the far left medium graymolecules, the Leu 10 region molecules, and the far right medium grayregion indicating a 10–100 fold reduction, and the black regions at Phe49 and Gly 7 indicating a >100 fold reduction in affinity for IGFBP-1.The black regions to the right of the detergent molecule correspond tothe symmetry-related IGF-1 molecule that forms the crystallographicdimer. The circle near Gln 15 indicates the C10 atom of the detergent,as noted above for FIG. 7A. This figure was prepared using the programINSIGHT (MSI, San Diego, Calif.).

FIG. 8 shows a graph resulting from a detergent/IGFBP competitionbinding study. In this experiment,N,N-bis(3-D-gluconamidopropyl)-deoxycholamine was used as a competitiveinhibitor of IGF-1 binding to immobilized IGFBP-1 (open circles) orIGFBP-3 (open squares). As a positive control, soluble IGFBP-1 (solidcircles) or IGFBP-3 (solid squares) was used as a competitive inhibitorof IGF-1 binding to immobilized IGFBP-1 or IGFBP-3, respectively. Eachdata point represents the average of three independent experiments.

FIG. 9A shows a non-linear least-squares analysis of sedimentationequilibrium data for IGF-1 in solution. Data collected at rotor speedsof 30,000 rpm (open triangles) and 35,000 rpm (open squares) were fit asan ideal monomer-dimer self-association model. The solid lines are thefits of the data. FIG. 9B shows the residuals plotted for both rotorspeeds after accounting for the data by the fitting procedure. They arerandomly distributed around zero, indicating that the monomer-dimermodel is correct for this interaction.

FIG. 10A shows a ribbon diagram determined by NMR of a complex of IGF-1and N,N-bis(3-D-gluconamidopropyl)-deoxycholamine), and FIG. 10B shows aribbon diagram determined by NMR of a complex of IGF-1 bound to aphage-derived IGF-1 antagonist peptide designated IGF-F1-1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. DEFINITIONS

As used herein, “IGF-1” refers to human insulin-like growth factor-1unless otherwise noted, and has the human native mature IGF-1 sequencewithout a N-terminal methionine, as described, for example, by EP230,869 published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; orEP 288,451 published Oct. 26, 1988.

An “IGFBP” or an “IGF binding protein” refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-1,whether or not it is circulatory (i.e., in serum or tissue). Thisdefinition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5,IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) orendothelial cell-specific molecule (ESM-1), as well as other proteinswith high homology to IGFBPs. Mac 25 is described, for example, inSwisshelm et al., Proc. Natl. Acad. Sci. USA, 92: 4472–4476(1995) and Ohet al., J. Biol. Chem., 271: 30322–30325(1996). PSF is described inYamauchi et al., Biochemical Journal, 303: 591–598 (1994). ESM-1 isdescribed in Lassalle et al., J. Biol. Chem., 271: 20458–20464 (1996).For other identified IGFBPs, see, e.g., EP 375,438 published 27 Jun.1990; EP 369,943 published 23 May 1990; U.S. Pat. No. 5,258,287; WO89/09268 published 5 Oct. 1989; Wood et al., Molecular Endocrinology, 2:1176–1185 (1988); Brinkman et al., The EMBO J., 7: 2417–2423 (1988); Leeet al., Mol. Endocrinol., 2: 404–411 (1988); Brewer et al., BBRC, 152:1289–1297 (1988); EP 294,021 published 7 Dec. 1988; Baxter et al., BBRC,147: 408–415 (1987); Leung et al., Nature, 330: 537–543 (1987); Martinet al., J. Biol. Chem., 261: 8754–8760 (1986); Baxter et al., Comp.Biochem. Physiol., 91B: 229–235 (1988); WO 89/08667 published 21 Sep.1989; WO 89/09792 published 19 Oct. 1989; and Binkert et al., EMBO J.,8: 2497–2502 (1989). IGFBP-1 and IGFBP-3 bind to different residues ofIGF-1.

As used herein, “human IGF-1 receptor” or just “IGF-1 receptor” refersto any receptor for IGF-1 found in humans and includes the Type 1 andType 2 IGF receptors in humans to which human IGF-1 binds, such as theplacental IGF-1R, etc.

An “indirect agonist of IGF-1” is a molecule that releases IGF-1 in situfrom IGFBP-3 or IGFBP-1 so that the IGF-1 released is active andinteracts with its receptor.

“Peptides” are molecules having at least two amino acids and includepolypeptides having at least about 60 amino acids. Preferably, thepeptides have about 10 to about 60 amino acids, more preferably about10–25, and most preferably about 12–25 amino acids. The definitionincludes linear and cyclic peptides, peptide derivatives, their salts,or optical isomers.

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic, and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, sheep,pigs, cows, etc. The preferred mammal herein is a human. The term“non-adult” refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented.

A “disorder” is any condition that would benefit from treatment with anIGF-1 agonist (“agonist disorder”) or antagonist (“antagonistdisorder”). This includes chronic and acute disorders or diseasesincluding those pathological conditions that predispose the mammal tothe disorder in question. The disorder being treated may be acombination of two or more of the agonist or antagonist disorders listedbelow.

Non-limiting examples of antagonist disorders include benign andmalignant tumors, leukemias and lymphoid malignancies, neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders, and inflammatory, angiogenic andimmunologic disorders, diabetic complications such as diabeticretinopathies or neuropathies, age-related macular degeneration,ophthalmic surgery such as cataract extraction, a corneal transplant,glaucoma filtration surgery and keratoplasty, surgery to correctrefraction, i.e., a radial keratotomy, also in sclera macular holes anddegeneration, retinal tears, vitreoretinopathy, miscellaneous disorders,cataract disorders of the cornea such as the sequelae of radialkeratotomy, dry eye, viral conjunctivitis, ulcerative conjunctivitis,wounds such as corneal epithelial wounds, Sjogren's syndrome, retinaldisorders such as macular and retinal edema, vision-limited scarring,retinal ischemia, and proliferative vitreous retinopathy.

More preferably, such antagonist disorders include diabeticcomplications exacerbated by IGF-1, ischemic injury, and diseasesassociated with undesirable cell proliferation such as cancer,restenosis, and asthma. If the disorder is a diabetic complicationexacerbated by IGF-1, such complication can include diabetic retinopathyor diabetic nephropathy. The efficacy of the treatment can be evidencedby a reduction in clinical manifestations or symptoms, including, forexample, improved renal clearance, improved vision, or a reduction inthe amount of IGF-1 available for binding to an IGF-1 receptor. If thedisorder is an ischemic injury, it can include strokes, myocardialischemia, and ischemic injury to the kidneys.

Examples of agonist disorders for purposes herein include any conditionthat would benefit from treatment with an IGF-1, including but notlimited to, for example, lung diseases, hyperglycemic disorders as setforth below, renal disorders, such as acute and chronic renalinsufficiency, end-stage chronic renal failure, glomerulonephritis,interstitial nephritis, pyelonephritis, glomerulosclerosis, e.g.,Kimmelstiel-Wilson in diabetic patients and kidney failure after kidneytransplantation, obesity, GH-insufficiency, Turner's syndrome, Laron'ssyndrome, short stature, undesirable symptoms associated with aging suchas obesity and increased fat mass-to-lean ratios, immunologicaldisorders such as immunodeficiencies including decreased CD4 counts anddecreased immune tolerance or chemotherapy-induced tissue damage, bonemarrow transplantation, diseases or insufficiencies of cardiac structureor function such as heart dysfunctions and congestive heart failure,neuronal, neurological, or neuromuscular disorders, e.g., peripheralneuropathy, multiple sclerosis, muscular dystrophy, or myotonicdystrophy, and catabolic states associated with wasting caused by anycondition, including, e.g., trauma or wounding, or infection such aswith a bacterium or human virus such as HIV, wounds, skin disorders, gutstructure and function that need restoration, and so forth. Thepreferred agonist disorders targeted for treatment herein are diabetesand obesity, heart dysfunctions, AIDS-related wasting, kidney disorders,neurological disorders, whole body growth disorders, and immunologicaldisorders.

As used herein, the term “hyperglycemic disorders” refers to all formsof diabetes and disorders resulting from insulin resistance, such asType I and Type II diabetes, as well as severe insulin resistance,hyperinsulinemia, and hyperlipidemia, e.g., obese subjects, andinsulin-resistant diabetes, such as Mendenhall's Syndrome, WernerSyndrome, leprechaunism, lipoatrophic diabetes, and other lipoatrophies.The preferred hyperglycemic disorder is diabetes, especially Type I andType II diabetes. “Diabetes” itself refers to a progressive disease ofcarbohydrate metabolism involving inadequate production or utilizationof insulin and is characterized by hyperglycemia and glycosuria.

“Biologically active” IGF-1 refers to IGF-1 that exhibits a biologicalproperty conventionally associated with an IGF-1 agonist or antagonist,such as a property that would allow treatment of one or more of thedisorders listed above.

The term “effective amount” refers to an amount of IGF-1 or apeptidomimetic or other compound, including chemical entities, effectiveto treat a disease or disorder in a mammal. In the case of cancer, forexample, the effective amount of the peptide may reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., slow to some extentand preferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow at least to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; promote apoptosis;and/or relieve to some extent one or more of the symptoms associatedwith the disorder.

A “precipitant” is an agent in a reservoir solution that precipitatesIGF-1 when mixed with an aqueous solution of IGF-1 and allowed toequilibrate so as to form IGF-1 crystals. Examples include chaotropicagents such as ammonium sulfate, polyethylene glycols (of a wide varietyof molecular weights ranging, for example, from about 2000 to 20,000),sodium citrate, sodium cacodylate, or a mixture thereof.

A “reservoir solution” is a solution of a precipitant and any otheringredient needed to provide IGF-1 crystals, for example, a detergentsuch as C₁₂E₉ (nonaethylene glycol monododecyl ether, nonaethyleneglycol monolauryl ether, polyoxyethylene (9) ether), C₁₂E₈ (octaethyleneglycol monododecyl ether, octaethylene glycol monolauryl ether,polyoxyethylene (8) lauryl ether), dodecyl-beta-D-maltopyranoside,lauric acid sucrose ester, cyclohexyl-pentyl-beta-D-maltoside,nonaethylene glycol octylphenol ether, cetyltrimethylammonium bromide,decyl-beta-D-maltopyranoside, lauryidimethylamine oxide,cyclohexyl-pentyl-beta-D-maltoside, n-dodecylsulfobetaine,3-(dodecyldimethylammonio)propane-1-sulfonate,nonyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, OSG,N,N-dimethyldecylamine-beta-oxide,methyl-6-O-(N-heptylcarbamoyl)alpha-D-glycopyranoside, sucrosemonocaproylate, heptyl-beta-D-thioglucopyranoside,octyl-beta-D-glucopyranoside, cyclohexyl-propyl-beta-D-maltoside,cyclohexylbutanoyl-N-hydroxyethyleglucamide, n-decylsulfobetaine,3-(decyidimethylammonio)propane-1-sulfonate, octanoyl-N-methylglucamide,hexyl-beta-D-glucopyranoside, andN,N-bis(3-D-gluconamidopropyl)-deoxycholamine. Preferably, the detergentis N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.

“Recrystallization” refers to the procedure, after the initial crystalsare grown and determined not to be very large or useful, of adding asubstance to the crystals, such as methyl pentanediol, which has theeffect of dissolving the crystals, but not diluting anything else muchin the crystallization mixture. Then over the course of several days, asthe crystallization droplet re-equilibrates with its reservoir solution,the crystals regrow, but this time much larger and more well ordered.

The term “associating with” refers to a condition of proximity betweenIGF-1 and a chemical entity, or portions thereof. The association may benon-covalent, wherein the juxtaposition is energetically favored byhydrogen bonding, van der Waals interaction, or electrostaticinteraction, or it may be a covalent association.

The term “binding site” refers to any or all of the sites where achemical entity binds or associates with IGF-1.

The term “structural coordinates” refers to the coordinates derived frommathematical equations related to the patterns obtained on diffractionof a monochromatic beam of x-rays by the atoms (scattering centers) of amolecule in crystal form. The diffraction data can be used to calculatean electron density map of the repeating units of the crystal. Thoseskilled in the art will understand that the data obtained are dependentupon the particular system used, and hence, different coordinates may infact describe the same crystal if such coordinates define substantiallythe same relationship as those described herein. An electron density mapmay be used to establish the positions of the individual atoms withinthe unit cell of the crystal.

Those of skill in the art understand that a set of structuralcoordinates determined by x-ray crystallography is not without standarderror. Appendix 1 shows the atomic coordinates of IGF-1. For the purposeof this invention, any set of structural coordinates of IGF-1 that havea root mean square deviation of equivalent protein backbone atoms ofless than about 2 Å when superimposed—using backbone atoms—on thestructural coordinates in Appendix 1 shall be considered identical.Preferably, the deviation is less than about 1 Å and more preferablyless than about 0.5 Å.

The term “heavy-atom derivatization” refers to a method of producing achemically modified form of a crystallized IGF-1. In practice, a crystalis soaked in a solution containing heavy metal atom salts, ororganometallic compounds, e.g., lead chloride, gold thiomalate,thimerosal, or uranyl acetate, which can diffuse through the crystal andbind to the surface of the protein. The location of the bound heavymetal atom(s) can be determined by x-ray diffraction analysis of thesoaked crystal. This information can be used to generate the phaseinformation used to construct the three-dimensional structure of themolecule.

The term “unit cell” refers to a basic shaped block. The entire volumeof a crystal may be constructed by regular assembly of such blocks. Eachunit cell comprises a complete representation of the unit of pattern,the repetition of which builds up the crystal.

The term “space group” refers to the arrangement of symmetry elements ofa crystal.

The term “molecular replacement” refers to a method that involvesgenerating a preliminary structural model of a crystal whose structuralcoordinates are unknown, by orienting and positioning a molecule whosestructural coordinates are known, e.g., the IGF-1 coordinates inAppendix 1, within the unit cell of the unknown crystal, so as to bestaccount for the observed diffraction pattern of the unknown crystal.Phases can then be calculated from this model, and combined with theobserved amplitudes to give an approximated Fourier synthesis of thestructure whose coordinates are unknown. This in turn can be subject toany of the several forms of refinement to provide a final accuratestructure of the unknown crystal. (See, e.g., Lattman, E., “Use of theRotation and Translation Functions,” Methods in Enzymology, 115: 55–77(1985); Rossman, ed., “The Molecular Replacement Method,” Int. Sci. Rev.Ser. No. 13 (Gordon and Breach: New York, 1972)). Using the structuralcoordinates of IGF-1 provided by this invention, molecular replacementmay be used to determine the structural coordinates of a crystallineco-complex, unknown ligand, mutant, or homolog, or of a differentcrystalline form of IGF-1. Additionally, the claimed crystal and itscoordinates may be used to determine the structural coordinates of achemical entity that associates with IGF-1.

The term “chemical entity” or “compound” as used herein means anymolecule, molecular complex, compound, peptidomimetic, or fragmentthereof that is not IGF-1. Preferably it is a molecule with high oralbioavailability, such as an organic chemical molecule, or a peptide.

B. MODES FOR CARRYING OUT THE INVENTION

The following detailed description of the invention encompasses thecrystal structure of IGF-1, methods of making an IGF-1 crystal, andmethods of using an IGF-1 crystal and its structural coordinates.

a. Crystal Structure of IGF-1

The claimed invention provides crystals of IGF-1 as well as thestructure of IGF-1 determined therefrom. Specifically, the claimedinvention provides crystals of IGF-1 having approximately the followingdimensions: a=31.831 Å, b=71.055 Å, c=65.995 Å, α=β=γ=90.000°. It has asymmetry, or space group, of C222₁. The ribbon structure thereof isshown in FIG. 2 having three helices, with the N-terminal B-regioncorresponding to residues 3–28, the C-region from residues 29–34, astretch of poorly ordered residues from residues 35–40, and the A-regionfrom residues 42–62. The D-region (residues 63–70) is essentiallydisordered. FIGS. 4 and 7 show that the detergent used in thecrystallization binds into a small hydrophobic cleft at the base of theB-helix of the structure. The IGF-1 can form a dimer in the crystal, asshown in FIG. 5, wherein the two tails are positioned at the dimerinterface. The buried surface area is 689 Å²/monomer, which is 1378 Å²total. The residues important for IGF-1R binding cluster at the dimerinterface, as shown in FIG. 6.

The characteristics of the claimed IGF-1 crystal are further describedin the Example herein and the structural coordinates thereof areprovided in Appendix 1.

b. Methods of Making an IGF-1 Crystal

In various embodiments, the claimed invention relates to methods ofpreparing crystalline forms of IGF-1 by first providing an aqueoussolution comprising IGF-1. A reservoir solution comprising a precipitantis then mixed with a volume of the IGF-1 solution and the resultantmixed volume is then crystallized. In a preferred step the crystals areagain dissolved and recrystallized. An example of a reagent that can beused for recrystallization is methyl pentanediol, which is preferred.The crystals are typically dissolved with this reagent in a small amountto minimize dilution effects of the other reagents and left to regrowfor a period of time. In an optional step, the crystalline IGF-1 isisolated from the mixed volume. Preferably, the IGF-1 is obtained from aprokaryotic cell, more preferably a bacterial cell, most preferably E.coli. Preferably it is secreted into the periplasm and prepared asdescribed in U.S. Pat. No. 5,723,310.

The concentration of IGF-1 in the aqueous solution may vary, but ispreferably about 1 to 50 mg/ml, more preferably about 5 to 15 mg/ml.Similarly, precipitants used in the invention may vary, and may beselected from any precipitant known in the art. Preferably, theprecipitant is selected from the group consisting of sodium citrate,ammonium sulfate, polyethylene glycol, sodium cacodylate, or a mixturethereof. More preferably the precipitant is polyethylene glycol bufferedwith sodium citrate or sodium cacodylate. Any concentration ofprecipitant may be used in the reservoir solution; however, it ispreferred that the concentration be about 20 to 25% if polyethyleneglycol, and about 1 to 10 M if sodium citrate, ammonium sulfate, orsodium cacodylate. Preferably, the reservoir solution further comprisesa detergent. Preferably, the detergent is present in an amount of about10 to 50 mM. Also preferably the detergent is N,N-bis(3-D-gluconamidopropyl)-deoxycholamine. The pH of the reservoirsolution may also be varied, preferably between about 4 to 10, mostpreferably about 6.5.

One skilled in the art will understand that each of these parameters canbe varied without undue experimentation and acceptable crystals willstill be obtained. In practice, once the appropriate precipitatingagents, buffers, or other experimental variables are determined for anygiven growth method, any of these methods or any other methods can beused to grow the claimed crystals. One skilled in the art can determinethe variables depending upon one's particular needs.

Various methods of crystallization can be used in the claimed invention,including vapor diffusion, batch, liquid-bridge, or dialysiscrystallization. Vapor diffusion crystallization is preferred. See, e.g.McPherson et al., Preparation and Analysis of Protein Crystals, Glick,ed. (John Wiley & Co., 1982), pp. 82–159; Jancarik et al., J. Appl.Crystallogr., 24: 409–411 (1991).

In vapor diffusion crystallization, a small volume (i.e., a fewmilliliters) of protein solution is mixed with a solution containing aprecipitant. This mixed volume is suspended over a well containing asmall amount, i.e. about 1 ml, of precipitant. Vapor diffusion from thedrop to the well will result in crystal formation in the drop.

The dialysis method of crystallization utilizes a semipermeablesize-exclusion membrane that retains the protein but allows smallmolecules (i.e. buffers and precipitants) to diffuse in and out. Indialysis, rather than concentrating the protein and the precipitant byevaporation, the precipitant is allowed to slowly diffuse through themembrane and reduce the solubility of the protein while keeping theprotein concentration fixed.

The batch methods generally involve the slow addition of a precipitantto an aqueous solution of protein until the solution just becomesturbid; at this point the container can be sealed and left undisturbedfor a period of time until crystallization occurs.

Thus, applicants intend that the claimed invention encompass any and allmethods of crystallization. One skilled in the art can choose any ofsuch methods and vary the parameters such that the chosen method resultsin the desired crystals.

The most preferred method of crystallization involves the method whereinthe IGF-1, after isolation from the cell and formulation in, forexample, an acetate, citrate, or succinate buffer, as described, forexample, in U.S. Pat. No. 5,681,814 and WO 99/51272, is optionallydesalted if necessary to a pH of about 4–5, preferably about 4.5, toform an aqueous solution. Then, a droplet of the aqueous solution ismixed with about 24% polyethylene glycol buffered to about pH 6.5 witheither about 0.1M sodium citrate or about 0.1M sodium cacodylate andwith about 1 μl of about 1.4 mMN,N-bis(3-D-gluconamidopropyl)-deoxycholamine as detergent. Thissolution is then equilibrated by vapor diffusion crystallization withabout 1 mL of about 24% polyethylene glycol buffered to about pH 6.5with either about 0.1M sodium citrate or about 0.1M sodium cacodylateuntil crystallization droplets are formed, usually about 4–5 days. Thenabout 2 μl of about 100% methyl pentanediol is added to thecrystallization droplets so as to dissolve the crystals overnight andthereby form new crystals, usually within a week's time.

The crystal structure was determined by combined anomalous scatteringfrom intrinsic sulfur and fortuitous bromide ion as discussed in detailin the Example below.

c. Methods of Using an IGF-1 Crystal and its Coordinates

The crystalline IGF-1 herein can be used for various purposes. Forexample, the crystallization process itself further purifies the IGF-1to homogeneity. Thus, one such purpose is to provide a highly purifiedIGF-1 that can be used as a standard or control in a diagnostic setting,for example, as a molecular weight marker, or as an ELISA, radioassay,or radioreceptor assay control. Moreover, crystalline IGF-1 is stable atroom temperature, can be lyophilized readily, and is less apt to degradethan less pure compositions.

In another use for the invention herein, crystals of IGF-1 of a size andquality to allow performance of x-ray diffraction studies enable thoseof skill in the art to conduct studies relating to the bindingproperties of IGF-1, as well as the binding properties of IGFBPs, IGF-1receptors, and ALS that associate with the IGF-1.

Furthermore, structural information derived from a peptide crystalstructure can be used for the identification of chemical entities, forexample, small organic and bioorganic molecules such as peptidomimeticsand synthetic organic molecules that bind IGF-1 and preferably block orprevent an IGF-1-mediated or -associated process or event, or that actas IGF-1 agonists. An exemplary approach to such a structure-basedcompound design is described in Structure Based Drug Design, PandiVeerapandian, ed. (Marcell Dekker: New York 1997).

By way of example, having determined the three-dimensional structure ofthe IGF-1, the skilled artisan constructs a model of the IGF-1 such asthose depicted in FIGS. 2 and 5. Since every atom of a peptide orpolypeptide can be depicted as a sphere of the appropriate van der Waalsradius, a detailed surface map of the folded IGF-1 can be constructed.The surface that results is known as the van der Waals surface. The“solvent-accessible surface” is the surface that is accessible to achemical probe, a water molecule herein, and is constructed by rolling awater molecule of appropriate radius on the outside of the peptidemaintaining contact with the van der Waals surface. Those parts of thevan der Waals surface that contact the surface of the water moleculedefine a continuous surface known as the “solvent-accessible surface.”(Creighton, Thomas E., Proteins: structure and molecular properties,2nd. ed. (W. H. Freeman and Company, 1984), pp 227–229).

Such chemical entities presenting a solvent-accessible surface thatmimics the solvent-accessible surface of the IGF-1 can be constructed bythose skilled in the art. By way of example, the skilled artisan cansearch three-dimensional structural databases of compounds to identifythose compounds that position appropriate functional groups in similar3-dimensional structural arrangement, then build combinatorial chemistrylibraries around such chemical entities to identify those with highaffinity.

One approach enabled by this invention is the use of the structuralcoordinates of IGF-1 to design chemical entities that bind to orassociate with IGF-1 and alter the physical properties of the chemicalentities in different ways. Thus, properties such as, for example,solubility, affinity, specificity, potency, on/off rates, or otherbinding characteristics may all be altered and/or maximized.

One may design desired chemical entities by probing an IGF-1 crystalwith a library of different entities to determine optimal sites forinteraction between candidate chemical entities and IGF-1. For example,high-resolution x-ray diffraction data collected from crystals saturatedwith solvent allows the determination of where each type of solventmolecule adheres. Small molecules that bind tightly to those sites canthen be designed and synthesized and tested for the desired activity.Once the desired activity is obtained, the molecules can be furtheraltered to maximize desirable properties.

The invention also contemplates computational screening ofsmall-molecule databases or designing of chemical entities that can bindin whole or in part to IGF-1. They may also be used to solve the crystalstructure of mutants, co-complexes, or the crystalline form of any othermolecule homologous to, or capable of associating with, at least aportion of IGF-1.

One method that may be employed for this purpose is molecularreplacement. An unknown crystal structure, which may be any unknownstructure, such as, for example, another crystal form of IGF-1, an IGF-1mutant or peptide, or a co-complex with IGF-1, or any other unknowncrystal of a chemical entity that associates with IGF-1 that is ofinterest, may be determined using the structural coordinates as setforth in Appendix 1. Co-complexes with IGF-1 may include, but are notlimited to, IGF-1-IGFBP-3, IGF-1-IGFBP-3-ALS, IGF-1-receptor,IGF-1-peptide, or IGF-1-small molecule. This method will provide anaccurate structural form for the unknown crystal far more quickly andefficiently than attempting to determine such information without theinvention herein.

The information obtained can thus be used to obtain maximally effectiveinhibitors or agonists of IGF-1. The design of chemical entities thatinhibit or agonize IGF-1 generally involves consideration of at leasttwo factors. First, the chemical entity must be capable of physically orstructurally associating with IGF-1. The association may be anyphysical, structural, or chemical association, such as, for example,covalent or noncovalent bonding, or van der Waals, hydrophobic, orelectrostatic interactions.

Second, the chemical entity must be able to assume a conformation thatallows it to associate with IGF-1. Although not all portions of thechemical entity will necessarily participate in the association withIGF-1, those non-participating portions may still influence the overallconformation of the molecule. This in turn may have a significant impacton the desirability of the chemical entity. Such conformationalrequirements include the overall three-dimensional structure andorientation of the chemical entity in relation to all or a portion ofthe binding site.

The potential inhibitory or binding effect of a chemical entity on IGF-1may be analyzed prior to its actual synthesis and testing by the use ofcomputer-modeling techniques. If the theoretical structure of the givenchemical entity suggests insufficient interaction and associationbetween it and IGF-1, the need for synthesis and testing of the chemicalentity is obviated. However, if computer modeling indicates a stronginteraction, the molecule may then be synthesized and tested for itsability to bind to IGF-1. Thus, expensive and time-consuming synthesisof inoperative compounds may be avoided.

An inhibitory or other binding compound of IGF-1 may be computationallyevaluated and designed by means of a series of steps in which chemicalentities or fragments are screened and selected for their ability toassociate with the individual binding sites of IGF-1.

Thus, one skilled in the art may use one of several methods to screenchemical entities or fragments for their ability to associate withIGF-1. This process may begin by visual inspection of, for example, thebinding site on a computer screen based on the IGF-1 coordinates inAppendix 1. Selected fragments or chemical entities may then bepositioned in a variety of orientations, or “docked,” within anindividual binding pocket of IGF-1. Docking may be accomplished usingsoftware such as Quanta and Sybyl, followed by energy minimization andmolecular dynamics with standard molecular mechanics force fields, suchas CHARMM and AMBER.

Specialized computer programs may be of use for selecting interestingfragments or chemical entities. These programs include, for example,GRID, available from Oxford University, Oxford, UK; MCSS or CATALYST,available from Molecular Simulations, Burlington, Mass.; AUTODOCK,available from Scripps Research Institute, La Jolla, Calif.; DOCK,available from University of California, San Francisco, Calif., andXSITE, available from University College of London, UK.

Once suitable chemical entities or fragments have been selected, theycan be assembled into an inhibitor or agonist. Assembly may be by visualinspection of the relationship of the fragments to each other on thethree-dimensional image displayed on a computer screen, in relation tothe structural coordinates disclosed herein.

Alternatively, one may design the desired chemical entities de novo,experimentally, using either an empty binding site or optionallyincluding a portion of a molecule with desired activity. Thus, forexample, one may use solid-phase screening techniques where either IGF-1or a fragment thereof, or candidate chemical entities to be evaluated,are attached to a solid phase, thereby identifying potential binders forfurther study.

Basically, any molecular modeling techniques may be employed inaccordance with the invention; these techniques are known, or readilyavailable to those skilled in the art. It will be understood that themethods and compositions disclosed herein can be used to identify,design, or characterize not only entities that will associate or bind toIGF-1, but alternatively to identify, design, or characterize entitiesthat, like IGF-1, will bind to the receptor, thereby disrupting theIGF-1-receptor interaction. The claimed invention is intended toencompass these methods and compositions broadly.

Once a compound has been designed or selected by the above methods, theefficiency with which that compound may bind to IGF-1 may be tested andmodified for the maximum desired characteristic(s) using computationalor experimental evaluation. Various parameters can be maximizeddepending on the desired result. These include, but are not limited to,specificity, affinity, on/off rates, hydrophobicity, solubility, andother characteristics readily identifiable by the skilled artisan.

Additionally, the invention is useful for the production ofsmall-molecule drug candidates. Thus, the claimed crystal structures maybe also used to obtain information about the crystal structures ofcomplexes of the IGF-1 and small-molecule inhibitors. For example, ifthe small-molecule inhibitor is co-crystallized with IGF-1, then thecrystal structure of the complex can be solved by molecular replacementusing the known coordinates of IGF-1 for the calculation of phases. Suchinformation is useful, for example, for determining the nature of theinteraction between the IGF-1 and the small-molecule inhibitor, and thusmay suggest modifications that would improve binding characteristicssuch as affinity, specificity, and kinetics.

d. Other Methods

The invention herein is also useful in providing a method of identifyingindirect agonists of IGF-1 based on the inhibitory properties ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine with respect to IGFBPs.This method comprises the steps of: comparing the ability ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine to inhibit binding ofIGFBP-1 or -3 to IGF-1 with the ability of a candidate IGF-1 indirectagonist to inhibit such binding; and determining whether the candidateIGF-1 indirect agonist can inhibit such binding at least as well asN,N-bis(3-D-gluconamidopropyl)-deoxycholamine can so inhibit thebinding.

Preferably the comparison is accomplished by competition assay betweenN,N-bis(3-D-gluconamidopropyl)-deoxycholamine and the candidate IGF-1indirect agonist, using IC₅₀ to measure ability to inhibit IGFBPbinding. In a more preferred embodiment, inhibition of binding ismeasured by pre-incubating N,N-bis(3-D-gluconamidopropyl)-deoxycholamineor the candidate agonist molecule with IGF-1 expressed on bacteriophageparticles and measuring residual binding of IGF-1 to IGFBP-1 or IGFBP-3in a plate-based assay, such as an ELISA.

The invention further provides a method of identifying indirect agonistsof IGF-1 comprising co-crystallizing the candidate agonist with IGF-1 toform a co-crystalline structure and determining if the candidate agonistmolecule binds to one or both of two patches on IGF-1. The first patchcontains the amino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13,Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57, andthe second patch cohtains the amino acid residues Val 11, Gln 15, Phe23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55 of IGF-I (SEQ ID NO: 1).For purposes herein, binding means that there is at least one contactbetween each listed amino acid residue of a given patch and thecandidate agonist molecule that is less than or equal to 6 angstroms inthe co-crystalline structure. Such a candidate agonist molecule willhave the property of inhibiting binding of IGFBP-1 or IGFBP-3 to IGF-1.The preferred such candidate agonist molecule will inhibit binding ofIGFBP-1 or -3 to IGF-1 at least as well as N,N-bis(3-D-gluconamidopropyl)-deoxycholamine. More preferred is themethod wherein inhibition of binding is measured using a competitionassay between N, N-bis(3-D-gluconamidopropyl)-deoxycholamine and thecandidate agonist molecule. Most preferred is the method whereininhibition of binding is measured by pre-incubating N,N-bis(3-D-gluconamidopropyl)-deoxycholamine or the candidate agonistmolecule with IGF-1 expressed on bacteriophage particles and measuringresidual binding of IGF-1 to IGFBP-1 or IGFBP-3 in a plate-based ELISAassay.

The N,N-bis(3-D-gluconamidopropyl)-deoxycholamine detergent herein canbe used as a template to perform design of small-molecule drugs thatelicit the same effect as the detergent (compete with IGF-1 for IGFBPbinding and subsequent disruption of the interaction of IGFBP with IGF-1to free IGF-1 in situ so that it is active and will interact with thereceptor. As opposed to the other detergents tested in the Examplesbelow, N,N-bis(3-D-gluconamidopropyl)-deoxycholamine lacks an oxygenatom at position C10. This region of the detergent is in close contactwith the side-chain atoms of residues Leu 5, Leu 54, and Leu 57 ofIGF-1. Molecules with this same type of conformation would work asindirect IGF-1 agonists.

The indirect agonist so identified can be used in a method for treatingan agonist disorder wherein an effective amount of the indirect agonistof IGF-1 is administered to a mammal with such a disorder. Hence, suchagonist may be used therapeutically in a pharmaceutical preparation, forexample, in clinical trials or commercialized for the agonist disordersas defined herein. Thus, the formulation of the indirect agonist hereincan be used to treat any condition that would benefit from treatmentwith IGF-1, including, for example, diabetes, chronic and acute renaldisorders, such as chronic renal insufficiency, necrosis, etc., obesity,hyperinsulinemia, GH-insufficiency, Tumer's syndrome, short stature,undesirable symptoms associated with aging such as increasinglean-mass-to-fat ratios, immuno-deficiencies including increasing CD4counts and increasing immune tolerance, catabolic states associated withwasting, etc., Laron dwarfism, insulin resistance, and so forth.

For therapeutic use, the indirect agonist composition herein may bedirectly administered to the mammal by any suitable technique, includingorally, parenterally, intranasally, or intrapulmonarily, and can beadministered locally or systemically. The specific route ofadministration will depend, e.g., on the medical history of the patient,including any perceived or anticipated side or reduced effects usingIGF-1, and the disorder to be treated. Examples of parenteraladministration include subcutaneous, intramuscular, intravenous,intraarterial, and intraperitoneal administration. Most preferably, theadministration is by continuous infusion (using, e.g., minipumps such asosmotic pumps), or by injection (using, e.g., intravenous orsubcutaneous means). The administration may also be as a single bolus orby slow-release depot formulation. Most preferably, the direct agonistis administered orally or by infusion or injection, at a frequency of,preferably, one-half, once, twice, or three times daily, most preferablydaily.

The agonist composition to be used in the therapy will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with the agonist), the site of delivery of theagonist composition, the method of administration, the scheduling ofadministration, and other factors known to clinical practitioners. The“effective amount” of agonist for purposes herein is thus determined bysuch considerations and must be an amount that treats the disorder inquestion.

As a general proposition, the total pharmaceutically effective amount ofagonist administered parenterally per dose will be in the range of about1 μg/kg/day up to about 100 mg/kg/day, preferably 10 μg/kg/day up toabout 10 mg/kg/day. If given continuously, the agonist is generallyadministered in doses of about 1 μg/kg/hour up to about 100 μg/kg/hour,either by about 1–4 injections per day or by continuous subcutaneousinfusions, for example, using a minipump or a portable infusion pump. Anintravenous bag solution may also be employed. The key factor inselecting an appropriate dose is the result obtained as measured bycriteria as are deemed appropriate by the practitioner. If the agonistis administered together with insulin, the latter is used in loweramounts than if used alone, down to amounts which by themselves havelittle effect on blood glucose, i.e., in amounts of between about 0.1IU/kg/24 hour to about 0.5 IU/kg/24 hour.

For parenteral administration, in one embodiment, the agonist isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulation is prepared by contacting the agonistuniformly and intimately with a liquid carrier or a finely divided solidcarrier or both. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low-molecular-weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; nonionic surfactantssuch as polysorbates, poloxamers, or PEG; and/or neutral salts, e.g.,NaCl, KCl, MgCl₂, CaCl₂, etc.

The agonist is typically formulated individually in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, ata pH of about 4.5 to 8. The final formulation, if a liquid, ispreferably stored at a temperature of about 2–8° C. for up to about fourweeks. Alternatively, the formulation can be lyophilized and provided asa powder for reconstitution with water for injection that is stored asdescribed for the liquid formulation.

The agonist to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic agonistcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The agonist ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. The disclosures of all literature and patentcitations mentioned herein are expressly incorporated by reference.

EXAMPLE 1 Crystallization and Characterization of IGF-1 Crystals

Crystallization of IGF-1 and Data Collection

Recombinant human IGF-1 (rhIGF-1) was obtained as described in theExamples of U.S. Pat. No. 5,723,310 using a polymer/salt combination forphase-forming species and formulated as described in the Examples ofU.S. Pat. No. 5,681,814 (acetate, NaCl, polysorbate 20, and benzylalcohol). Specifically, the initial isolation of IGF-1 from E. coli wasachieved using aqueous two-phase separation (Hart et al.,Bio/Technology, 12: 1113–1117 (1994)), followed by refolding (Hart etal., Biotechnol. Appl. Biochem., 20: 217–232 (1994)), and subsequentchromatographic purification, including large-scale reverse-phasehigh-performance liquid chromatography (Olson et al., J. Chromatogr.,A675: 101–112 (1994)). It was placed in a vial containing 7 ml of 10mg/ml rhIGF-1. Prior to crystallization, the IGF-1 was desalted into0.15 M NaCl and 20 mM sodium acetate (pH 4.5), and diluted to a finalconcentration of 10 mg/ml. Initially, crystallization trials were set upin the presence of 1 mM of an IGF-1-binding peptide. However, no peptidewas ever observed in the crystal, and crystals grown in the absence ofthe peptide were later shown to be isomorphous to the specimen reportedhere. A 4-μl droplet of the IGF-1 solution was mixed with 5 μl ofreservoir solution (24% polyethylene glycol 3350 buffered to pH 6.5 with0.1M sodium cacodylate) and 1 μl of 14 mM ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine, which is obtained in aCRYSTAL SCREEN™ reagent kit used for crystallization conditionscreenings and available from Hampton Research, Inc., Laguna Nigel,Calif. This solution was allowed to equilibrate via vapor diffusion(Jancarik et al., supra) with 1 ml of reservoir solution. Thus, a dropof the mixture was suspended under a plastic cover slip over thereservoir solution. Small crystals with a thin, plate-like morphologyappeared within 4–5 days. At this point, 2 μl of 100% methyl pentanediol(MPD) (to a final concentration of 20%) was added to the crystallizationdroplet, and the crystals dissolved overnight. Within 1 week, crystalsreappeared and grew to final dimensions of 0.2 mm×0.1 mm×0.05 mm withnoticeably sharper edges. These crystals were used for all subsequentanalysis.

Those of skill in the art will appreciate that the aforesaidcrystallization conditions can be varied. By varying the crystallizationconditions, other crystal forms of IGF-1 may be obtained. Suchvariations may be used alone or in combination, and include, forexample, varying final protein concentrations between about 5 and 35mg/ml; varying the IGF-1-to-precipitant ratio, varying precipitantconcentrations between about 20 and 30% for polyethylene glycol, varyingpH ranges between about 5.5 and 7.5, varying the concentration or typeof detergent, varying the temperature between about −5 and 30° C., andcrystallizing IGF-1 by batch, liquid bridge, or dialysis methods usingthe above conditions or variations thereof. See McPherson et al. (1982),supra.

Characterization of IGF-1 Crystals

A single crystal was transferred from the mother liquor to acryo-protectant solution consisting of 25% (w/v) polyethylene glycol3350, 30% MPD, 0.2 M sodium cacodylate pH 6.5, 2.8 mM ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine, and 1 M NaBr. Thediffraction was to 1.8 Å. After 30 seconds in this solution, the crystalwas flash-cooled by plunging it into liquid nitrogen. The technique offreezing the crystals essentially immortalizes them and produces a muchhigher quality data set. All subsequent manipulations and x-ray datacollection were performed at 100° Kelvin.

A 4-wavelength MAD data set was collected at beamline 9–2 at theStanford Synchrotron Radiation Laboratory, with the order of the datasets as follows: Br peak (λ1), low-energy remote (λ2), Br inflection(λ3), and high-energy remote (λ4). The Br peak and inflection pointswere estimated from fluorescence scans of the crystal, and thelow-energy remote was chosen to be 1.54 angstroms, to maximize the smallsulfur anomalous signal at this wavelength while minimizing absorptioneffects. No inverse beam geometry was used. Data reduction was performedusing Denzo and Scalepack (Otwinowski and Minor, Methods in Enzymology,276: 307–326 (1997)). To determine the most accurate scale and B-factorspossible, data for all four wavelengths were initially scaled together,assuming no anomalous signal. The scale and B-factors determined fromthis scaling run were then applied to each of the four data sets.

The crystals belong to space group C222₁ with unit cell dimensions orconstants of: a=31.83 Å, b=71.06 Å, and c=66.00 Å. α=β=γ=90.000°. Theasymmetric unit of the crystals contained a monomer of IGF-1 bound to asingle detergent molecule, yielding a Matthew's coefficient of 2.4Å³/Da, or 48.1% solvent. The solvent content of the crystals was about55%.

Structure Determination

Initial attempts at determining the structure of IGF-1 by molecularreplacement, using either the available NMR models of IGF-1 or thecrystal structure of insulin, were unsuccessful. For this reason, thestructure was determined de novo by Br multiwavelength anomalousdispersion (MAD) (Dauter et al., Acta Crystallogr. D56: 232–237 (2000)).

The coordinates of the single-bound bromide were determined by manualinspection of the anomalous and dispersive difference Patterson maps.The hand ambiguity was resolved by phase refinement using the programSHARP (De La Fortelle and Bricogne, Methods in Enzymology, 276: 472–494(1997)) from Global Phasing Limited, 43 Newton Road, Cambridge CB2 2AL,ENGLAND, followed by examination of anomalous-difference Fourier mapscalculated using the λ2 Bijvoet differences. A cluster of six peaks forone hand of the Br coordinates was consistent with the disulfidestructure of insulin (PDB entry: IZNI). These six peaks correspond tothe six Cys Sγatoms in IGF-1; a seventh sulfur (Met 59 Sδ) was neverdetected in anomalous-difference Fourier maps, presumably due to itshigher temperature factor (36.7 Å²). At this point, the six CysSγpositions were included in the phase refinement, with the λ1 data setused as a reference. Throughout the phase refinement, the Br f″ wasrefined for the λ1 data set, f′ and f″ were refined for λ3, and bothwere kept fixed for data sets λ2 and λ4; the f″ and f′ values for sulfurwere kept fixed at the theoretical values for each wavelength. The smallanomalous signal from the sulfur atoms had a modest effect on thephasing statistics, but the resulting electron-density maps showedimproved connectivity, especially in the less well ordered regions ofIGF-1.

Density modification (solvent flattening and histogram mapping) wasperformed using DM (Collaborative Computational Project Number 4, ActaCrystallogr., D50: 760–763 (1994); Cowtan, Joint CCP4 and ESF-EACBMNewsletter on Protein Crystallography, 31: 34–38 (1994)), and theresulting electron-density maps were of high quality. Approximately 50%of the structure, corresponding to the three helical regions of IGF-1,was built directly into the experimental electron-density maps using theprograms O (Jones et al., Acta Crystallogr., A47: 110–119 (1991)) andQUANTA (version 97.0, MSI, San Diego, Calif.). Several rounds of phasecombination using Sigmaa (Collaborative Computational Project Number 4,supra; Read, Acta Crystallogr., A42: 140–149 (1986)) allowed theremainder of the molecule to be modeled. Atomic positional andrestrained B-factor refinement utilized the maximum-likelihood targetfunction of CNX (Brünger et al., Acta Crystallogr., D54: 905–921 (1998)and MSI, San Diego, Calif.), coupled with a “mask”-type bulk solventcorrection and anisotropic overall B-factor scaling.

The final model contains residues 3–34 and 41–64 of IGF-1, oneN,N-bis(3-D-gluconamidopropyl)-deoxycholamine molecule, 1 Br⁻, and 50water molecules. The model was refined against the λ3 data set, sincethe data statistics demonstrated this data set to be of higher qualitythan the others. All data from 20- to 1.8-angstrom resolution wereincluded in the refinement, with no application of a sigma cutoff.Secondary structure assignments were made with the program PROMOTIF(Jones et al., supra; Hutchinson and Thornton, Protein Science, 5:212–220 (1996)).

While the well-ordered positions of IGF-1 were essentially identicalusing the two sets of phases, the more flexible regions of the moleculeshowed dramatically improved connectivity upon inclusion of the sulfursin the phasing. Experimental electron density maps showing the turnregion of IGF-1 immediately following the first helix (residues 19, 20,and 21) indicate that using the combined Br and S phases resulted in amuch more well-connected map than using just the Br phases alone. Atthis point, using the Br+S phases, about 50% of the molecule could betraced directly into the experimental maps.

Description of the Structure

After several cycles of model building and phase combination, the finalmodel, shown in FIG. 2, contains residues 3–34 and 41–64 of IGF-1 (SEQID NO: 1), a single-bound detergent molecule, and 46 water molecules.The R factor to 1.8 Å is 23.7%, and the free R factor is 26.9%, withgood stereochemistry. The N-terminal B-region corresponds to residues3–28, the C-region from 29–34, a stretch of poorly ordered residues from35–40, and the A-region from 42–62 of IGF-I (SEQ ID NO: 1). The D-region(63–70) is essentially disordered.

The structure of IGF-1 is similar to insulin (see FIG. 3), with aRoot-Mean-Squared-Deviation (RMSD) of 3 Å over backbone atoms that areconserved between the two molecules. Most of these deviations occur inthe flexible regions, and when only the helical regions are considered,the RMSD between alpha-carbon atoms is about 0.47 Å. The majordifference is the extension of the C-region, for which there is nocounterpart in mature insulin, away from the body of the molecule. Thisloop contains many of the residues that are known to be important forreceptor binding.

An extensive alanine-scan mutagenesis study on IGF-1 has shown whichresidues are important for binding to IGFBP-1 and IGFBP-3 (Dubaquie andLowman, supra). The residues that bind to IGFBP-3 are similar to thosethat bind IGFBP-1, although IGFBP-3 is believed to depend more onbackbone interactions and is less severely affected by alaninemutations. There is no one dramatic spot where residues important forIGFBP-1 and IGFBP-3 binding are clustered, and mutations that impairbinding are scattered all over the molecule. There appears to be aslight clustering of sites at the N-terminus, with many of these sitesbeing intrinsically hydrophobic.

As shown in FIGS. 4 and 7, the detergent molecule binds into a smallhydrophobic cleft at the base of the B-helix. There are several directside-chain contacts to the detergent from residues 5, 7, and 10. Despitethe overlap of the detergent binding site with a portion of theIGFBP-1/IGFBP-3 binding epitope, the preliminary results suggest,without being limited to any one theory, that the detergent does notinhibit binding of these proteins to IGF-1. The opposite face of thedetergent is making a symmetry contact to the opposite face of IGF-1.

As shown in FIG. 5, there is only one large crystal packing contactbetween symmetry-related IGF-1 molecules, which results in a symmetrichomodimer. The buried surface area is 1378 Å², which is in the range ofphysiologically relevant protein-protein interfaces.

FIG. 6 shows that the residues known to be important for receptorbinding cluster at this dimer interface. Shown are Tyr24, Thr29, Tyr31,and Tyr60. Mutation of these residues results in anywhere from 6–20×loss in affinity for receptor for individual mutations, or 240→1200×loss in affinity for double mutations. Also shown are Phe23 and Phe25,which are interchangeable with Phe24 and Tyr26 of insulin, with no lossof affinity.

Further Description of the Structure

IGF-1 (SEO ID NO: 1) is composed primarily of three helical segmentscorresponding to the B-helix (IGF-1 residues 7–18) and two A-helices(IGF-1 residues 43–47 and 54–58) of insulin. The hydrophobic core isessentially identical to that described for the NMR structures of IGF-1,including the three disulfide linkages between Cys 6 and Cys 48, Cys 18and Cys 61, and Cys 47 and Cys 52, as noted in the references above.Residues 3 through 6 do not form any regular secondary structure, andhence, the structure described herein can be classified as being mostsimilar to the T-form of insulin (Derewenda et al., Nature, 338: 594–596(1989)). Indeed, when IGF-1 and the T-form of insulin are superimposedon the Cα positions of their respective helical segments (IGF-1 SEQ IDNO: 1) residues 8–19, 42–49, and 54–61; insulin (SEO ID NO: 3) residuesB9–B20, A1–A8, and A13–A20) the RMSD is only 0.47 angstroms. As ininsulin, residues 18–21 at the end of the B-helix form a type II′β-turn,which redirects the backbone from the B-helix into an extended region.Residues 24–27 form a type VIII β-turn to accommodate the C-region,which extends away from the core of IGF-1, and interacts with asymmetry-related molecule. Residues 30–33 form a well-defined type IIbeta-turn, prominently displaying Tyr 31 at the i+1 position. Residues35–40 have not been modeled, as the electron density in this region isweak and disconnected. Only the first two residues of the D–region(residues 63 and 64) are ordered in the structure.

The C-region of IGF-1 (SEQ ID NO: 1) mediates a two-fold symmetriccrystal-packing interaction across the α-axis of the unit cell. Thisinteraction buries 689 Å² of solvent-accessible surface area from eachmolecule of IGF-1, or 1378 Å² total, and is the largest interface in thecrystal. A total of 28 intermolecular contacts of distance 3.6 Åor lessare formed via this interface, with the next most extensive crystalpacking interaction forming only nine contacts. The core of theinterface is dominated by Tyr24 and Pro28 from each monomer, which bury39 Å² and 57 Å² of solvent-accessible surface area, respectively. Thearomatic ring of Tyr 31, which lies at the tip of the loop at thefurthest point from the core of IGF-1, packs against the phenolic ringsof Phe 23 and Phe 25 of the symmetry-related molecule. In addition tothese hydrophobic interactions, two main-chain hydrogen bonds (Tyr 31N-Phe 23 O and Ser 34 N-Asp 20 O of IGF-I, SEQ ID NO: 1) are present inthe dimer interface. Residues from the D-region (62–64) are alsopartially sequestered by this dimer formation. Because of theseinteractions, most of the C-region in the crystal is well-ordered,providing the first high-resolution view of the conformation of thisbiologically important loop.

Although 72 detergent compounds, including the similar3-((3-cholamidopropyl) dimethylammonio)-1-propane sulphonate (CHAPS) and3-((3-cholamidopropyl)dimethylammonio)-2-hydroxypropanesulfonic acid(CHAPSO) detergents, were screened in crystallization trials, only N,N-bis(3-D-gluconamidopropyl)-deoxycholamine yielded crystals. A singlemolecule of N, N-bis(3-D-gluconamidopropyl)-deoxycholamine interactswith residues, forming a small hydrophobic cleft on one surface of IGF-1(SEQ ID NO: 1 (Leu 5, Phe 16, Val 17, Leu 54, and Leu 57) (FIG. 7A). Thepreference for N, N-bis(3-D-gluconamidopropyl)-deoxycholamine isexplained, without being limited to any one theory, by the absence of anoxygen atom at position C10 in the detergent molecule. This region ofthe detergent is in close contact with the side chain atoms of residuesLeu 5, Leu 54, and Leu 57 in IGF-1. The opposite face of the detergentmediates a symmetry contact with residues Val 11, Leu 14, and Gln 15 ofa symmetry-related IGF-l molecule. Intriguingly, this face of N,N-bis(3-D-gluconamidopropyl)-deoxycholamine also contacts the edge ofthe dimer interface, with close contacts to Phe 23 and Phe 25 of thesame IGF-1 molecule, as well as Tyr 31 and Gly 32 of the dimeric partner(FIG. 7B). A more detailed analysis indicates that the detergent bindsto two patches of binding pockets of IGF-1 (SEQ ID NO: 1). One patch hasthe amino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13, Phe 16, Val17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57, and the secondpatch has the amino acid residues Val 11, Gin 15, Phe 23, Phe 25, Asn26, Val 44, Phe 49, and Arg 55. Binding is defined by having at leastone contact between each listed amino acid residue and the candidateagonist molecule that is less than or equal to 6 angstroms.

Discussion

The C-region in the IGF-1 crystal structure extends out from the core ofthe molecule, with residues 30–33 forming a canonical type II beta-turn,and the remainder of the C-region forming a crystallographic dimer witha symmetry-related molecule. Tyr 31 has been implicated as being acritical determinant for IGF-1R binding (Bayne et al. (Vol. 264), supra;Bayne et al. (Vol. 265), supra; Cascieri et al., supra), and itslocation at the tip of this extension places it in an ideal location tointeract with a receptor molecule. While this region of IGF-1 is notwell-defined by NMR data, the conformation of the C-region in thecrystal is likely to reflect a prevalent solution conformation. There isevidence of a reverse turn at the tip of the loop and a hinge bending atthe loop termini of IGF-2 (SEQ ID NO: 2; Torres et al., supra). Thus,while crystal packing forces undoubtedly help stabilize the orientationof this loop, its conformation appears to be consistent with thesolution structure of the closely related IGF-2.

The size of the interface formed by the crystallographic dimer is wellwithin the range of buried surface area in known biological complexes(Janin and Chothia, J. Biol. Chem., 264: 16027–16030 (1990)). Inaddition, this interaction partially excludes from solvent several ofthe residues known to be important for binding to the IGF-1R, includingPhe 23 (69% buried), Tyr 24 (64% buried), Phe 25 (29% buried), and Tyr31(38% buried) of IGF-I, SEQ ID NO: 1. Other groups have also reportedhomodimeric interactions of IGF-1 (SEQ ID NO: 1) and IGF-2 (SEQ ID NO:2). Laajoki et al., (2000), supra, report that at a concentration of 1mM, an engineered form of IGF-1 (Long-[Arg³]IGF-1) partitions into about20% dimer/80% monomer, a ratio that is in good agreement with theestimate of 3.6 mM K_(d). In their NMR study of IGF-2, Torres et al.,supra, reported that the amide protons of residues in the C-region wereslowly exchanging with solvent, suggesting that IGF-2 forms a homodimerin solution. However, despite the significant amount of surface areathat is buried upon dimer formation in the crystal, the affinity ofIGF-1 for itself is very weak. In addition, the known bindingstoichiometry of one IGF-1 molecule per receptor dimer (De Meyts, supra)makes it difficult to rationalize the biological significance of IGF-1dimerization. In conclusion, the IGF-1 dimer in this crystal formresults from the high concentration of IGF-1 in the crystallizationexperiment, and does not represent a physiologically relevant form ofthe molecule.

The very low quality of NMR spectroscopic data obtained for IGF-1 atnear-neutral pH has been attributed to a combination of self-associationand internal mobility that leads to a large variation in resonance linewidth (Cooke et al., supra). As a result, NOESY spectra acquired onIGF-1 contain many broad, overlapped peaks and few sharp well-resolvedcorrelations. NOESY spectra collected for IGF-1 in the presence of anexcess of N, N-bis(3-D-gluconamidopropyl) -deoxycholamine have a similarappearance. Thus, detergent binding is not sufficient to eliminate theaggregation or inherent flexibility of IGF-1 and does not facilitatecharacterization of the solution conformation of the protein. Likewise,detergent binding does not alter the aggregation state of IGF-1, asassessed by analytical ultracentrifugation experiments in the presenceof N, N-bis(3-D-gluconan-iidopropyl)-deoxycholamine. This is in contrastto observations in the crystalline state where addition of N,N-bis(3-D-gluconamidopropyl)-deoxycholamine leads to a well-packedcrystallographic dimer and crystals that diffract to high resolution.Jansson et at, J. Biol. Chem., 273: 24701–24707 (1998) noted that thelack of NMR assignments in the region immediately surrounding Cys 6 ofIGF-I, SEQ ID NO: 1, which includes Leu 5 and Gly 7, was indicative ofthe Cys 6-Cys 48 disulfide undergoing intermediate exchange between acis and trans configuration. The fact that the detergent binds to oneface of the B-helix immediately opposite this disulfide suggests,without being limited to any one theory, that it may serve to stabilizethis region of the molecule by more complete packing of the hydrophobiccleft. Indeed, in the crystal structure herein, the Cys 6-Cys 48 isclearly in the trans conformation, and there is no evidence of multipleconformations.

Conclusion

The crystal structure of IGF-1 has been determined using anomalousscattering from the intrinsic sulfur atoms and a Br-ion bound at afortuitous halide-binding site. The structure is very similar toinsulin, with the only major difference being the C-region, whichprotrudes from the body of the protein and mediates a homodimericinteraction. The amount of buried surface area is consistent with thefact that at neutral pH, IGF-1 undergoes self-association in aconcentration-dependent manner. In addition, several residues that areimportant for receptor binding are found at this dimer interface,suggesting, without being limited to any one theory, that effects onreceptor binding by mutation of these residues may be a result ofdisruption of the dimer, rather than direct contact with the receptorsurface.

EXAMPLE 2

Diffusion-based Measurement of Detergent Binding

NMR-derived diffusion measurements were used to estimate the K_(d) forthe interaction between IGF-1 andN,N-bis(3-D-gluconamidopropyl)-deoxycholamine. Samples were prepared in50 mM phosphate buffer in D₂0, pH 6.5 (uncorrected meter reading), andcontained: 1.0 mM N,N-bis(3-D-gluconamidopropyl)-deoxycholamine+0.5 mMIGF-1, 0.5 mM N, N-bis(3-D-gluconamidopropyl)-deoxycholamine+0.25 mMIGF-1; 0.25 mM N,N-bis(3-D-gluconamidopropyl)-deoxycholamine+0.125 mMIGF-1; or N,N-bis(3-D-gluconamidopropyl)-deoxycholamine only (1.0, 0.5,or 0.25 mM). All spectra were acquired at 40° C. on a Bruker AVANCE 500™spectrometer (Bruker Analytik GmbH) equipped with a 5-mm triple-axisgradient, triple-resonance probe. Diffusion measurements were made witha bipolar pulse pair method with δ=5 ms, τ=2 ms, and Δ=25 or 40 ms forN,N-bis(3-D-gluconamidopropyl)-deoxycholamine alone orN,N-bis(3-D-gluconamidopropyl)-deoxycholamine+IGF-1, respectively (Wu etal., J. Magn. Reson., Ser. A 115: 260–264 (1995)). Spectra werecollected with 128 to 1024 transients as the z-gradient strength wasincreased from 0.009 to 0.45 T·m⁻¹ in 18 equal increments; measurementswere made at least twice on each sample. Spectra were processed and peakheights extracted with the program FELIX (v98.0, MSI, San Diego).Diffusion constants, proportion of bound detergent, and resulting K_(d)were extracted as described by Fejzo et al., Chemistry & Biology, 6:755-769 (1999). Spectra were also collected on samples containing 1.0 mM3-((3-cholamidopropyl)dimethylammonio)-1-propane sulphonate, azwitterionic detergent used for membrane solubilization, and 1.0 mM3-(3-cholamidopropyl)dimethylammonio)-1-propane sulphonate+0.5 mM IGF-1.Two-dimensional NOESY spectra (Jeener et al., J. Chem. Phys., 71:4546–4553 (1979)) were collected on a 0.5-mM sample of IGF-1 in thepresence or absence of 1.0 mMN,N-bis(3-D-gluconamidopropyl)-deoxycholamine with a mixing time of 100ms.

IGF-1 Phage ELISA

E. coli cells (XL1-Blue, Stratagene) freshly transformed with the phagevector pIGF-g3 displaying human IGF-1 as described in Dubaquie andLowman, supra, were grown overnight in 5 ml of 2YT medium (Sambrook etal., Molecular Cloning: A Laboratory Handbook (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989)). The phage particlesdisplaying IGF-1 were titered against IGFBP-1 and IGFBP-3 to obtain a500–1000-fold dilution for preincubation with serial dilutions of thedetergents and binding protein standards for 45 minutes. Microwell clearpolystyrene immunoplates with a MAXISORP™ surface (Nunc, Denmark) werecoated with IGFBP-1 or IGFBP-3 protein overnight at 4° C. (50 μl at 3μg/mL in 50 mM carbonate buffer, pH 9.6), blocked with 0.5% TWEEN® 20polyoxyethylene sorbitan monolaurate (Atlas Chemical Co.), and PBS andwashed eight times with PBS and 0.05% TWEEN® 20 polyoxyethylene sorbitanmonolaurate. The samples were added to the plates for 30 minutes. Plateswere washed eight times with PBS and 0.05% TWEEN® 20 polyoxyethylenesorbitan monolaurate, incubated with 50 μL of 1:10,000 horseradishperoxidase/anti-M13 antibody conjugate (Amersham Pharmacia Biotech,Piscataway, N.J.) in PBS and 0.5% BSA for 30 minutes, and then washedeight times with PBS and 0.05% TWEEN® 20 polyoxyethylene sorbitanmonolaurate and two times with PBS. Plates were developed using atetramethylbenzidine substrate (Kirkegaard and Perry, Gaithersburg,Md.), stopped with 1.0 H₃P0₄, and read spectrophotometically at 450 nm.

Sedimentation Equilibrium Analysis

The self-association of IGF-1 was determined by sedimentationequilibrium analysis. The experiments were conducted at 20° C. in anOPTIMA™ XL-A/XL-1 analytical ultracentrifuge (Beckman Coulter, Inc.).The samples were prepared in 0.1 M citrate buffer, pH 6.5, 75 mM NaClwith a loading concentration from 1 mM to 0.01 mM. The concentrationgradients were measured at rotor speeds of 25000 and 30000 rpm at 280 nmor 285 nm using a scanning absorption optical system. The attainment ofan equilibrium state was verified by comparing successive scans afterapproximately 16 hours. The partial specific volume of IGF-1 wascalculated from its amino acid composition. The data were fit as asingle ideal species or the ideal dimer self-association models using anon-linear least-squares fitting program, NONLIN (Johnson et al.,Biophys. J., 36: 578–588(1981)). The association constants weredetermined from the best-fit values of the model, returned by non-linearleast-squares regression.

Results:

N,N-bis(3-D-gluconamidopropyl)-deoxycholamine Binds to IGF-1 inSolution.

The affinity of IGF-1 for3-((3-cholamidopropyl)dimethylammonio-1-propane sulphonate andN,N-bis(3-D-gluconamidopropyl)-deoxycholamine was ascertained usingsolution-NMR methods. The chemical shift changes observed during atitration of N,N-bis(3-D-gluconamidopropyl)-deoxycholamine into a 0.5 mMIGF-1 solution suggested that the affinity was submillimolar and noteasily measurable from such data. Instead, diffusion measurements weremade on samples at varying IGF-1 concentrations containing 2 molarequivalents of detergent and also on several samples of detergent alone(the detergent concentration was always less than the critical micelleconcentration of 1.4 mM forN,N-bis(3-D-gluconamidopropyl)-deoxycholamine and 14 mM for3-((3-cholamidopropyl)dimethylammonio)-1-propane sulphonate). Thedecrease in diffusion constant of the detergent in the presence of theprotein can be used to estimate the proportion of detergent bound to theprotein (Fejzo et al., supra). Since the total concentration ofdetergent and protein is known, a value of the dissociation constant canbe determined. At the three protein concentrations studied (0.5 mM, 0.25mM, and 0.125 mM), K_(d) values of 220, 440, and 430 μM were obtained,respectively. This technique has routinely been applied to smallmolecules (several hundred Daltons molecular weight or less) binding tolarge proteins. In this particular case, the ligand is relatively large(862 Da) and the protein is relatively small (7648 Da); hence, thedifferential decrease in diffusion constant on binding is small. Thisincreases the uncertainty with which the dissociation constant can bemeasured. Given this, the data described above suggest that the K_(d)for the interaction betweenN,N-bis(3-D-gluconamidopropyl)-deoxycholamine and IGF-1 is 300±150 μM. Asimilar analysis of the (3-(3-cholamidopropyl)dimethylammonio)-1-propanesulphonate diffusion data suggests that that K_(d) in this case isgreater than 3 mM.

N,N-bis(3-D-gluconamidopropyl)-deoxycholamine Blocks IGFBP-1 and IGFBP-3Binding.

To examine the binding epitope ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine on IGF-1, the detergentwas preincubated with IGF-1 expressed on bacteriophage particles, andthe level of residual binding to IGFBP-1 and IGFBP-3 was measured in aplate-based assay (ELISA). As a control, soluble IGFBP-1 was alsotested. As shown in FIG. 8,N,N-bis(3-D-gluconamidopropyl)-deoxycholamine inhibited IGF-1 on phagefrom binding to IGFBP-1 and IGFBP-3 with IC₅₀ values of 480±170 μM and275±152 μM, respectively. These numbers must be interpretedconservatively, however, since the critical micelle concentration ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine (1.4 mM) presents an upperlimit on the curve in FIG. 8. In contrast to the effect ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine, the closely relateddetergent 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulphonatedid not show any inhibition of binding at any of the concentrationstested up to 1 mM. Despite the limitations of the experiment, the IC₅₀values obtained for N,N-bis(3-D-gluconamidopropyl)-deoxycholamine are ingood agreement with the NMR-based estimate of a K_(d) of ˜300 μM for theN,N-bis(3-D-gluconamidopropyl)-deoxycholamine-IGF-1 interaction.

Self-Association of IGF-1.

The sedimentation equilibrium data show that IGF-1 undergoesself-association in solution. The average molecular weight increasedwith increasing protein concentration from 0.01 mM to 1 mM. The averagemolecular weight at the highest concentration studied (1 mM) is about37% higher than the monomer molecular weight (10.4 KDa at 1 mM versus7.6 KDa monomer molecular weight). At concentrations below 0.05 mM, noself-association was observed, and IGF-1 exists only as a monomer insolution at neutral pH. If it is assumed that thehigher-molecular-weight species are IGF-1 dimers, the sedimentation datacan be fit as a monomer-dimer model with a K_(d) of 3.6±1.0 mM (FIG. 9).

Discussion

Several studies have identified residues in IGF-1 (SEQ ID NO: 1) thatare important for IGFBP binding (Clemmons et al., Endocrinology, 131:890–895 (1992); Dubaquie and Lowman, supra; Jansson et al., supra; Oh etal., (1993), supra; Lowman et al., (1998), supra; and Dubaquie et al.,Endocrinology ,142: 165–173 (2001)). Dubaquie and Lowman, supra,identified two distinct patches on IGF-1 that interact with IGFBP-1 andIGFBP-3. Patch I consists of Glu 7, Leu 10, Val 11, Leu 14, Phe 25, Ile43, and Val 44, while patch 2 consists of Glu 3, Thr 4, Leu 5, Phe 16,Val 17, and Leu 54. In the crystal structure of IGF-1, these two patchesare involved in detergent-mediated crystal packing contacts.(Specifically, Patch 1 of the crystal structure of IGF-1 consists ofamino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13, Phe 16, Val 17,Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57, and Patch 2 of thecrystal structure of IGF-1 consists of amino acid residues Val 11, Gln15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Mg 55, wherein bindingoccurs if there is at least one contact between each listed amino acidresidue and the candidate agonist molecule that is less than or equal to6 angstroms.)

The overlap of the detergent binding site with the IGFBP interactionsurfaces is entirely consistent with the observation herein thatN,N-bis(3-D-gluconamidopropyl)-deoxycholamine blocks IGFBP-1 and IGFBP-3binding. In contrast, N,N-bis(3-D-gluconamidopropyl)-deoxycholamine doesnot inhibit IGF-1R-mediated signaling in a cell-based receptoractivation assay. These results are consistent with prior studies thatdemonstrated different binding epitopes on IGF-1 for receptor and IGFBPinteractions (Bayne et al., supra, (Vol. 264); Bayne et al., supra,(Vol. 265); Cascieri et al., supra). The identification ofN,N-bis(3-D-gluconamidopropyl)-deoxycholamine as an inhibitor of IGFBPinteractions allows the ability to develop small-molecule drugs orpeptidomimetics that disrupt the IGF-1/IGFBP complex in vivo, therebyreleasing receptor-active IGF-1 from the systemic, inactive pool. Suchdrugs include orally bioavailable therapy for metabolic disease such asdiabetes.

Recently, Zeslawski et al. (EMBO J., 20: 3638–3644 (2001) published thecrystal structure of IGF-1 in complex with the N-terminal domain ofIGFBP-5. The structure of that complex is entirely consistent with themodel of detergent inhibition of IGFBP binding presented herein, andalso disclosed by Vajdos et al., Biochemistry, 40: 11022–11029 (2001).The NMR determination of a complex of IGF-1 bound to a phage-derivedIGF-1 antagonist peptide designated IGF-F1-1 (RNCFESVAALRRCMYG (SEQ IDNO:4)), in comparison with other IGF-1 crystal structures, shows that,without limitation to any one theory, a portion of the A-chain (helixIII) is mobile in solution, and adopts slightly different conformationswhen bound to different ligands (detergent, peptide, binding protein).

The complex between peptide IGF-F1-1 and IGF-1 was determined from NMRspectroscopy data collected at 600 and 800 MHz. IGF-1 uniformly labeledwith ¹³C and ¹⁵N was prepared using the scheme outlined by Reilly andFairbrother, J. Biomol. NMR, 4: 459–462 (1994) and purified according tothe protocol in Vajdos et al., supra. A slight molar excess of unlabeledIGF-F1-1 was mixed with a 1.5 mM solution of ¹³C/¹⁵N IGF-1 and ¹H, ¹³C,and ¹⁵N NMR resonances assigned from double- and triple-resonance NMRexperiments as described by Cavanagh et al. in Protein NMR Spectroscopy,Principles and Practice (Academic Press: New York, 1996). Distancerestraints within IGF-1 were identified from ¹³C-edited NOESY HSQCspectra and ¹⁵N-edited NOESY HSQC spectra (Cavanagh et al, supra).

Intermolecular restraints between IGF-1 and the peptide were obtainedfrom an ω1-filtered, ω2-edited ¹³C HSQC-NOESY spectrum (Lee et al., FEBSLett., 350: 87–90 (1994)). Intrapeptide distance restraints wereobtained from a 2-D ¹³C-filtered NOESY spectrum. In addition, φ dihedralangle restraints were obtained from an HNHA spectrum (Cavanagh et al.,supra), and χ1 restraints were derived from HNHB and short-mixing-timeTOCSY spectra (Clore et al., J. Biomolec. NMR, 1: 13–22(1991)).Additional φ, ψ restraints were obtained from an analysis of the H^(α),N, C^(α), C^(β), and CO chemical shifts using the program TALOS(Cornilescu et al., J. Biomol. NMR, 13: 289–302 (1999)).

In total, 899 distance restraints (779 intra-IGF-1; 33 intra-peptide; 87intermolecular), 16 hydrogen bond restraints in helix I, and 138dihedral angle restraints (71 Φ; 44 Ψ23 χ1) were used to generate anensemble of structures using a torsion-angle dynamics protocol with thecomputer program CNX (Accelrys Inc., San Diego). The structure of IGF-1(SEQ ID NO: 1) was well defined for the B-region (residues 2–25) and theA-region (residues 41–63) with a mean RMSD from the mean structure forbackbone heavy atoms of 0.32±0.06 Å. The C-region (26–40) and theD-region (62–70) were not well defined by the available data. The 20structures of lowest restraint violation energy had good backbonestereochemistry (80% of residues in the most favored region of Φ/ψ spacewith none in disallowed regions) and contained few violations of theexperimental restraints (mean maximum distance restraint violation 0.09±0.02 Å). IGF-F1-1 adopts a conformation very similar to that determinedfor the peptide by itself in solution. The conformation of IGF-1contains three helices (residues 7–18, 43–49, and 54–60) and is similarto that seen at lower resolution in previous NMR studies of uncomplexedIGF-1 (see e.g. Cooke et al., supra; Sato et al., supra; and Laajoki etal., supra).

FIG. 10 shows the comparison for the detergent and phage peptidecomplexes. Specifically, FIG. 10A shows a ribbon diagram of a complex ofIGF-1 and N,N-bis(3-D-gluconamidopropyl)-deoxycholamine), and FIG. 10Bshows a complex of IGF-1 bound to the phage-derived peptide IGF-F1-1.The B-region (helix I) adopts a very similar conformation in bothcomplexes. The C-loop is only partially ordered in the detergentcomplex, and ill defined in the peptide complex. Ligand-induceddifferences are observed for the A-region of IGF-1 (Helix III), at boththe backbone (residues 52–60) and side chain (leucine 54 and 57) level.Without limitation to any one theory, maleability in this A-region areais believed to be what allows IGF-1 to bind to so many proteins (sixIGFBPs and three receptors).

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the objectives of the presentinvention.

APPENDIX 1 REMARK 3 CRYSTAL STRUCTURE OF IGF-I SOLVED USING MAD REMARK 3REFINEMENT. REMARK 3 PROGRAM: X-PLOR(online) 98.1 REMARK 3 AUTHORS:BRUNGER REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTIONRANGE HIGH (ANGSTROMS): 1.80 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS):20.00 REMARK 3 DATA CUTOFF (SIGMA(F)): 0.2 REMARK 3 DATA CUTOFF HIGH(ABS(F)): 10000000.00 REMARK 3 DATA CUTOFF LOW (ABS(F)): 0.001000 REMARK3 COMPLETENESS (WORKING + TEST) (%): 94.8 REMARK 3 NUMBER OFREFLECTIONS: 6870 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT.REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE R VALUE TESTSET SELECTION: RANDOM REMARK 3 R VALUE (WORKING SET): 0.237 REMARK 3FREE R VALUE: 0.269 REMARK 3 FREE R VALUE TEST SET SIZE (%): 5.6 REMARK3 FREE R VALUE TEST SET COUNT: 382 REMARK 3 ESTIMATED ERROR OF FREE RVALUE: 0.014 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK3 TOTAL NUMBER OF BINS USED: 6 REMARK 3 BIN RESOLUTION RANGE HIGH (A):1.80 REMARK 3 BIN RESOLUTION RANGE LOW (A): 1.91 REMARK 3 BINCOMPLETENESS (WORKING + TEST) (%): 74.4 REMARK 3 REFLECTIONS IN BIN(WORKING SET): 826 REMARK 3 BIN R VALUE (WORKING SET): 0.343 REMARK 3BIN FREE R VALUE: 0.439 REMARK 3 BIN FREE R VALUE TEST SET SIZE (%): 5.5REMARK 3 BIN FREE R VALUE TEST SET COUNT: 48 REMARK 3 ESTIMATED ERROR OFBIN FREE R VALUE: 0.063 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMSUSED IN REFINEMENT. REMARK 3 PROTEIN ATOMS: 475 REMARK 3 NUCLEIC ACIDATOMS: 0 REMARK 3 HETEROGEN ATOMS: 62 REMARK 3 SOLVENT ATOMS: 102 REMARK3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): 25.1 REMARK 3MEAN B VALUE (OVERALL, A**2): 33.8 REMARK 3 OVERALL ANISOTROPIC B VALUE.REMARK 3 B11 (A**2): 0.00 REMARK 3 B22 (A**2): 0.00 REMARK 3 B33 (A**2):0.00 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 0.00 REMARK 3 B23(A**2): 0.00 REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESDFROM LUZZATI PLOT (A): 0.23 REMARK 3 ESD FROM SIGMAA (A): 0.16 REMARK 3LOW RESOLUTION CUTOFF (A): 20.00 REMARK 3 REMARK 3 CROSS-VALIDATEDESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A): 0.28REMARK 3 ESD FROM C-V SIGMAA (A): 0.18 REMARK 3 REMARK 3 RMS DEVIATIONSFROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A): 0.020 REMARK 3 BOND ANGLES(DEGREES): 2.0 REMARK 3 DIHEDRAL ANGLES (DEGREES): 23.2 REMARK 3IMPROPER ANGLES (DEGREES): 1.09 REMARK 3 REMARK 3 ISOTROPIC THERMALMODEL: RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS.RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2): 4.33; 4.00 REMARK 3MAIN-CHAIN ANGLE (A**2): 6.46; 4.00 REMARK 3 SIDE-CHAIN BOND (A**2):5.41; 8.50 REMARK 3 SIDE-CHAIN ANGLE (A**2): 8.14; 9.00 REMARK 3 REMARK3 NCS MODEL: NONE REMARK 3 REMARK 3 NCS RESTRAINTS. RMS SIGMA/WEIGHTREMARK 3 GROUP 1 POSITIONAL (A): NULL; NULL REMARK 3 GROUP 1 B-FACTOR(A**2): NULL; NULL REMARK 3 REMARK 3 PARAMETER FILE 1:MSI_XPLOR_TOPPAR/protein_rep.param REMARK 3 PARAMETER FILE 2:../parameter.element REMARK 3 PARAMETER FILE 3: ../cyc.par REMARK 3PARAMETER FILE 4: ../../../g2mz/param19.sol REMARK 3 TOPOLOGY FILE 1:/usr/prop/msi/980/data/xplor/toppar/tophcsdx.pro REMARK 3 TOPOLOGY FILE2: ../topology.element REMARK 3 TOPOLOGY FILE 3: ../cyc.top REMARK 3TOPOLOGY FILE 4: ../../../g2mz/toph19.sol REMARK 3 REMARK 3 OTHERREFINEMENT REMARKS: BULK SOLVENT MODEL USED SEQRES 1 A 100 GLU THR LEUCYS GLY ALA GLU LEU VAL ASP ALA LEU GLN SEQRES 2 A 100 PHE VAL CYS GLYASP ARG GLY PHE TYR PHE ASN LYS PRO SEQRES 3 A 100 THR GLY TYR GLY SERSER THR GLY ILE VAL ASP GLU CYS SEQRES 4 A 100 CYS PHE ARG SER CYS ASPLEU ARG ARG LEU GLU MET TYR SEQRES 5 A 100 CYS ALA PRO LEU HOH HOH HOHHOH HOH HOH HOH HOH HOH SEQRES 6 A 100 HOH HOH HOH HOH HOH HOH HOH HOHHOH HOH HOH HOH HOH SEQRES 7 A 100 HOH HOH HOH HOH HOH HOH HOH HOH HOHHOH HOH HOH HOH SEQRES 8 A 100 HOH HOH HOH HOH HOH HOH HOH HOH HOHSEQRES 1 B 3 CYC BR HOH SSBOND 1 CYS A 6 CYS A 48 SSBOND 2 CYS A 18 CYSA 61 SSBOND 3 CYS A 47 CYS A 52 CRYST1 31.830 71.074 66.014 90.00 90.0090.00 C 2 2 21 16 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX20.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.0000000.00000 SCALE1 0.031417 0.000000 0.000000 0.00000 SCALE2 0.0000000.014070 0.000000 0.00000 SCALE3 0.000000 0.000000 0.015148 0.00000REMARK FILENAME=“final.pdb” REMARK r = 0.237371 free_r = 0.26887 REMARKDATE: Jan. 30, 2001 14:51:37 created by user: mhu ATOM 1 CB GLU A 320.808 24.058 21.698 1.00 46.92 ATOM 2 CG GLU A 3 19.515 23.283 21.4991.00 61.66 ATOM 3 CD GLU A 3 18.281 24.029 22.025 1.00 70.74 ATOM 4 OE1GLU A 3 18.254 24.408 23.221 1.00 74.11 ATOM 5 OE2 GLU A 3 17.332 24.23721.238 1.00 75.50 ATOM 6 C GLU A 3 22.417 22.169 21.324 1.00 36.23 ATOM7 O GLU A 3 21.616 21.223 21.375 1.00 38.90 ATOM 8 N GLU A 3 21.62323.389 19.405 1.00 38.88 ATOM 9 CA GLU A 3 21.994 23.550 20.854 1.0040.66 ATOM 10 N THR A 4 23.696 22.040 21.647 1.00 31.38 ATOM 11 CA THR A4 24.213 20.754 22.059 1.00 24.88 ATOM 12 CB THR A 4 25.301 20.21721.051 1.00 23.86 ATOM 13 OG1 THR A 4 26.426 21.106 21.070 1.00 30.08ATOM 14 CG2 THR A 4 24.786 20.115 19.645 1.00 25.92 ATOM 15 C THR A 424.825 20.983 23.441 1.00 20.71 ATOM 16 O THR A 4 24.715 22.065 24.0361.00 19.97 ATOM 17 N LEU A 5 25.435 19.949 23.986 1.00 19.32 ATOM 18 CALEU A 5 25.976 20.054 25.319 1.00 15.87 ATOM 19 CB LEU A 5 25.300 19.03626.254 1.00 22.19 ATOM 20 CG LEU A 5 23.859 19.367 26.714 1.00 28.27ATOM 21 CD1 LEU A 5 23.229 18.123 27.479 1.00 23.45 ATOM 22 CD2 LEU A 523.916 20.612 27.594 1.00 25.44 ATOM 23 C LEU A 5 27.472 19.662 25.2521.00 19.16 ATOM 24 O LEU A 5 27.742 18.527 24.980 1.00 20.58 ATOM 25 NCYS A 6 28.352 20.591 25.572 1.00 19.74 ATOM 26 CA CYS A 6 29.809 20.32025.520 1.00 21.84 ATOM 27 C CYS A 6 30.485 20.754 26.790 1.00 16.04 ATOM28 O CYS A 6 29.990 21.602 27.535 1.00 18.05 ATOM 29 CB CYS A 6 30.44821.077 24.361 1.00 22.37 ATOM 30 SG CYS A 6 29.753 20.589 22.733 1.0032.91 ATOM 31 N GLY A 7 31.685 20.185 27.039 1.00 19.09 ATOM 32 CA GLY A7 32.397 20.613 28.217 1.00 17.15 ATOM 33 C GLY A 7 31.698 20.668 29.5361.00 18.61 ATOM 34 O GLY A 7 31.069 19.683 29.965 1.00 15.50 ATOM 35 NALA A 8 31.835 21.779 30.240 1.00 13.90 ATOM 36 CA ALA A 8 31.276 21.89731.553 1.00 15.72 ATOM 37 CB ALA A 8 31.630 23.239 32.150 1.00 21.73ATOM 38 C ALA A 8 29.715 21.774 31.498 1.00 14.12 ATOM 39 O ALA A 829.116 21.316 32.469 1.00 14.45 ATOM 40 N GLU A 9 29.145 22.263 30.4121.00 15.00 ATOM 41 CA GLU A 9 27.653 22.198 30.355 1.00 21.86 ATOM 42 CBGLU A 9 27.118 22.975 29.140 1.00 23.00 ATOM 43 CG GLU A 9 27.116 24.48129.346 1.00 33.94 ATOM 44 CD GLU A 9 26.424 25.279 28.204 1.00 53.09ATOM 45 OE1 GLU A 9 25.790 24.700 27.260 1.00 50.65 ATOM 46 OE2 GLU A 926.521 26.528 28.270 1.00 63.42 ATOM 47 C GLU A 9 27.200 20.732 30.2781.00 19.13 ATOM 48 O GLU A 9 26.180 20.376 30.831 1.00 15.61 ATOM 49 NLEU A 10 27.974 19.902 29.589 1.00 16.50 ATOM 50 CA LEU A 10 27.68818.488 29.435 1.00 19.32 ATOM 51 CB LEU A 10 28.637 17.845 28.384 1.0016.05 ATOM 52 CG LEU A 10 28.552 16.296 28.272 1.00 18.02 ATOM 53 CD1LEU A 10 27.080 15.875 27.864 1.00 17.22 ATOM 54 CD2 LEU A 10 29.45915.858 27.153 1.00 17.33 ATOM 55 C LEU A 10 27.850 17.812 30.788 1.0020.39 ATOM 56 O LEU A 10 27.030 16.968 31.173 1.00 13.49 ATOM 57 N VAL A11 28.924 18.126 31.531 1.00 16.73 ATOM 58 CA VAL A 11 29.089 17.55032.838 1.00 13.49 ATOM 59 CB VAL A 11 30.518 17.917 33.453 1.00 16.29ATOM 60 CG1 VAL A 11 30.636 17.370 34.882 1.00 19.22 ATOM 61 CG2 VAL A11 31.603 17.350 32.528 1.00 17.93 ATOM 62 C VAL A 11 28.006 18.02533.824 1.00 13.78 ATOM 63 O VAL A 11 27.532 17.272 34.668 1.00 11.61ATOM 64 N ASP A 12 27.599 19.280 33.717 1.00 14.34 ATOM 65 CA ASP A 1226.573 19.795 34.651 1.00 15.14 ATOM 66 CB ASP A 12 26.166 21.243 34.2901.00 14.66 ATOM 67 CG ASP A 12 26.960 22.337 34.970 1.00 27.52 ATOM 68OD1 ASP A 12 27.448 22.152 36.103 1.00 29.53 ATOM 69 OD2 ASP A 12 27.03123.422 34.314 1.00 31.00 ATOM 70 C ASP A 12 25.275 18.941 34.366 1.0011.41 ATOM 71 O ASP A 12 24.564 18.555 35.313 1.00 14.02 ATOM 72 N ALA A13 25.006 18.744 33.115 1.00 16.37 ATOM 73 CA ALA A 13 23.761 18.00232.720 1.00 18.28 ATOM 74 CB ALA A 13 23.558 18.014 31.189 1.00 15.65ATOM 75 C ALA A 13 23.803 16.590 33.235 1.00 17.95 ATOM 76 O ALA A 1322.833 16.073 33.788 1.00 15.70 ATOM 77 N LEU A 14 24.954 15.920 33.0651.00 11.92 ATOM 78 CA LEU A 14 25.077 14.560 33.611 1.00 15.01 ATOM 79CB LEU A 14 26.443 13.965 33.164 1.00 13.58 ATOM 80 CG LEU A 14 26.45713.638 31.695 1.00 15.59 ATOM 81 CD1 LEU A 14 27.939 13.345 31.250 1.0017.99 ATOM 82 CD2 LEU A 14 25.601 12.370 31.414 1.00 22.05 ATOM 83 C LEUA 14 24.944 14.495 35.098 1.00 15.56 ATOM 84 O LEU A 14 24.341 13.55035.626 1.00 18.96 ATOM 85 N GLN A 15 25.591 15.431 35.831 1.00 12.18ATOM 86 CA GLN A 15 25.494 15.426 37.273 1.00 14.03 ATOM 87 CB GLN A 1526.379 16.526 37.916 1.00 17.47 ATOM 88 CG GLN A 15 27.917 16.185 37.6731.00 23.70 ATOM 89 CD GLN A 15 28.863 16.851 38.656 1.00 32.76 ATOM 90OE1 GLN A 15 29.187 18.020 38.510 1.00 28.85 ATOM 91 NE2 GLN A 15 29.31416.095 39.658 1.00 29.69 ATOM 92 C GLN A 15 24.061 15.631 37.745 1.0018.29 ATOM 93 O GLN A 15 23.684 15.090 38.729 1.00 18.76 ATOM 94 N PHE A16 23.297 16.400 37.012 1.00 18.23 ATOM 95 CA PHE A 16 21.916 16.69237.438 1.00 17.04 ATOM 96 CB PHE A 16 21.438 17.935 36.706 1.00 18.66ATOM 97 CG PHE A 16 20.060 18.334 37.110 1.00 18.49 ATOM 98 CD1 PHE A 1619.874 18.960 38.310 1.00 23.45 ATOM 99 CD2 PHE A 16 18.959 17.94736.339 1.00 21.34 ATOM 100 CE1 PHE A 16 18.558 19.216 38.788 1.00 27.25ATOM 101 CE2 PHE A 16 17.646 18.190 36.789 1.00 21.50 ATOM 102 CZ PHE A16 17.457 18.829 38.020 1.00 24.95 ATOM 103 C PHE A 16 21.018 15.47937.110 1.00 15.52 ATOM 104 O PHE A 16 20.248 15.013 37.971 1.00 20.37ATOM 105 N VAL A 17 21.160 14.923 35.916 1.00 16.12 ATOM 106 CA VAL A 1720.338 13.761 35.490 1.00 17.94 ATOM 107 CB VAL A 17 20.392 13.59833.932 1.00 21.28 ATOM 108 CG1 VAL A 17 19.831 12.219 33.456 1.00 25.51ATOM 109 CG2 VAL A 17 19.619 14.737 33.295 1.00 22.15 ATOM 110 C VAL A17 20.720 12.454 36.182 1.00 21.98 ATOM 111 O VAL A 17 19.843 11.65536.556 1.00 24.05 ATOM 112 N CYS A 18 22.015 12.222 36.411 1.00 17.42ATOM 113 CA CYS A 18 22.430 10.964 37.039 1.00 20.54 ATOM 114 C CYS A 1822.420 10.998 38.579 1.00 26.67 ATOM 115 O CYS A 18 22.386 9.967 39.2391.00 24.96 ATOM 116 CB CYS A 18 23.841 10.565 36.505 1.00 16.00 ATOM 117SG CYS A 18 23.947 10.463 34.717 1.00 29.49 ATOM 118 N GLY A 19 22.46212.198 39.147 1.00 32.94 ATOM 119 CA GLY A 19 22.464 12.366 40.595 1.0031.76 ATOM 120 C GLY A 19 23.563 11.580 41.263 1.00 37.69 ATOM 121 O GLYA 19 24.730 11.631 40.869 1.00 40.61 ATOM 122 N ASP A 20 23.186 10.81542.276 1.00 39.20 ATOM 123 CA ASP A 20 24.150 10.009 42.989 1.00 47.44ATOM 124 CB ASP A 20 23.471 9.355 44.187 1.00 60.22 ATOM 125 CG ASP A 2023.633 10.169 45.437 1.00 73.16 ATOM 126 OD1 ASP A 20 24.132 11.31145.326 1.00 79.05 ATOM 127 OD2 ASP A 20 23.275 9.675 46.524 1.00 78.87ATOM 128 C ASP A 20 24.843 8.946 42.149 1.00 37.42 ATOM 129 O ASP A 2025.954 8.521 42.472 1.00 38.02 ATOM 130 N ARG A 21 24.213 8.484 41.0841.00 26.27 ATOM 131 CA ARG A 21 24.883 7.495 40.267 1.00 33.90 ATOM 132CB ARG A 21 23.930 6.931 39.223 1.00 36.79 ATOM 133 CG ARG A 21 22.5137.382 39.490 1.00 50.60 ATOM 134 CD ARG A 21 21.547 6.260 39.495 1.0046.09 ATOM 135 NE ARG A 21 21.068 5.971 38.157 1.00 44.95 ATOM 136 CZARG A 21 20.533 6.865 37.329 1.00 42.44 ATOM 137 NH1 ARG A 21 20.3978.132 37.692 1.00 49.97 ATOM 138 NH2 ARG A 21 20.103 6.474 36.138 1.0034.61 ATOM 139 C ARG A 21 25.951 8.301 39.565 1.00 36.17 ATOM 140 O ARGA 21 25.786 9.474 39.374 1.00 37.51 ATOM 141 N GLY A 22 27.056 7.70739.184 1.00 35.48 ATOM 142 CA GLY A 22 27.983 8.551 38.437 1.00 28.95ATOM 143 C GLY A 22 27.566 8.445 36.983 1.00 24.58 ATOM 144 O GLY A 2226.409 8.046 36.674 1.00 22.09 ATOM 145 N PHE A 23 28.465 8.786 36.0651.00 15.51 ATOM 146 CA PHE A 23 28.157 8.704 34.664 1.00 15.18 ATOM 147CB PHE A 23 27.665 10.080 34.127 1.00 20.92 ATOM 148 CG PHE A 23 28.53911.242 34.571 1.00 20.47 ATOM 149 CD1 PHE A 23 28.271 11.908 35.762 1.0024.30 ATOM 150 CD2 PHE A 23 29.644 11.596 33.815 1.00 20.21 ATOM 151 CE1PHE A 23 29.126 12.965 36.237 1.00 25.52 ATOM 152 CE2 PHE A 23 30.52012.636 34.261 1.00 20.52 ATOM 153 CZ PHE A 23 30.259 13.307 35.458 1.0022.56 ATOM 154 C PHE A 23 29.397 8.332 33.920 1.00 20.77 ATOM 155 O PHEA 23 30.484 8.463 34.494 1.00 24.08 ATOM 156 N TYR A 24 29.242 7.89132.673 1.00 20.54 ATOM 157 CA TYR A 24 30.396 7.562 31.821 1.00 25.88ATOM 158 CB TYR A 24 30.393 6.126 31.295 1.00 33.47 ATOM 159 CG TYR A 2429.706 5.125 32.115 1.00 31.26 ATOM 160 CD1 TYR A 24 30.356 4.551 33.2041.00 43.74 ATOM 161 CE1 TYR A 24 29.763 3.573 33.939 1.00 47.84 ATOM 162CD2 TYR A 24 28.422 4.680 31.785 1.00 41.08 ATOM 163 CE2 TYR A 24 27.8183.692 32.524 1.00 39.08 ATOM 164 CZ TYR A 24 28.492 3.151 33.589 1.0044.15 ATOM 165 OH TYR A 24 27.949 2.164 34.357 1.00 56.07 ATOM 166 C TYRA 24 30.332 8.362 30.588 1.00 24.71 ATOM 167 O TYR A 24 29.264 8.85930.221 1.00 31.49 ATOM 168 N PHE A 25 31.473 8.437 29.901 1.00 21.41ATOM 169 CA PHE A 25 31.546 9.091 28.625 1.00 18.28 ATOM 170 CB PHE A 2532.885 9.831 28.479 1.00 22.56 ATOM 171 CG PHE A 25 32.945 11.076 29.2941.00 22.04 ATOM 172 CD1 PHE A 25 33.336 11.040 30.625 1.00 22.70 ATOM173 CD2 PHE A 25 32.496 12.281 28.757 1.00 29.30 ATOM 174 CE1 PHE A 2533.264 12.190 31.432 1.00 25.64 ATOM 175 CE2 PHE A 25 32.415 13.44029.557 1.00 28.31 ATOM 176 CZ PHE A 25 32.794 13.394 30.888 1.00 27.30ATOM 177 C PHE A 25 31.385 8.046 27.534 1.00 20.94 ATOM 178 O PHE A 2530.992 8.341 26.411 1.00 21.90 ATOM 179 N ASN A 26 31.708 6.793 27.8681.00 25.83 ATOM 180 CA ASN A 26 31.598 5.697 26.899 1.00 27.61 ATOM 181CB ASN A 26 33.020 5.296 26.396 1.00 28.31 ATOM 182 CG ASN A 26 33.7376.469 25.737 1.00 29.50 ATOM 183 OD1 ASN A 26 34.438 7.267 26.395 1.0038.35 ATOM 184 ND2 ASN A 26 33.508 6.626 24.462 1.00 32.35 ATOM 185 CASN A 26 30.924 4.534 27.633 1.00 23.53 ATOM 186 O ASN A 26 31.274 4.21428.753 1.00 23.60 ATOM 187 N LYS A 27 29.958 3.879 27.025 1.00 26.93ATOM 188 CA LYS A 27 29.325 2.795 27.791 1.00 33.05 ATOM 189 CB LYS A 2728.018 2.384 27.113 1.00 34.53 ATOM 190 CG LYS A 27 27.014 3.521 27.0841.00 37.05 ATOM 191 CD LYS A 27 26.126 3.416 25.878 1.00 39.60 ATOM 192CE LYS A 27 24.768 4.078 26.139 1.00 42.35 ATOM 193 NZ LYS A 27 24.2444.674 24.865 1.00 39.82 ATOM 194 C LYS A 27 30.242 1.603 27.870 1.0031.68 ATOM 195 O LYS A 27 30.732 1.173 26.848 1.00 31.77 ATOM 196 N PROA 28 30.494 1.065 29.073 1.00 35.47 ATOM 197 CD PRO A 28 30.001 1.49130.392 1.00 34.96 ATOM 198 CA PRO A 28 31.388 −0.110 29.148 1.00 36.66ATOM 199 CB PRO A 28 31.468 −0.435 30.637 1.00 40.26 ATOM 200 CG PRO A28 30.367 0.359 31.287 1.00 40.80 ATOM 201 C PRO A 28 30.784 −1.25228.322 1.00 36.18 ATOM 202 O PRO A 28 29.558 −1.344 28.152 1.00 36.79ATOM 203 N THR A 29 31.614 −2.107 27.763 1.00 34.63 ATOM 204 CA THR A 2931.026 −3.155 26.945 1.00 41.92 ATOM 205 CB THR A 29 31.824 −3.39125.661 1.00 51.94 ATOM 206 OG1 THR A 29 32.938 −4.249 25.947 1.00 52.79ATOM 207 CG2 THR A 29 32.313 −2.071 25.086 1.00 55.75 ATOM 208 C THR A29 30.897 −4.498 27.635 1.00 35.36 ATOM 209 O THR A 29 30.068 −5.32227.238 1.00 34.58 ATOM 210 N GLY A 30 31.721 −4.720 28.643 1.00 33.36ATOM 211 CA GLY A 30 31.659 −5.997 29.323 1.00 33.97 ATOM 212 C GLY A 3032.109 −7.243 28.536 1.00 31.86 ATOM 213 O GLY A 30 32.227 −7.275 27.3051.00 34.94 ATOM 214 N TYR A 31 32.265 −8.325 29.275 1.00 23.05 ATOM 215CA TYR A 31 32.723 −9.576 28.690 1.00 25.72 ATOM 216 CB TYR A 31 33.144−10.505 29.813 1.00 21.15 ATOM 217 CG TYR A 31 34.274 −9.992 30.633 1.0024.03 ATOM 218 CD1 TYR A 31 34.066 −9.497 31.892 1.00 17.01 ATOM 219 CE1TYR A 31 35.106 −8.997 32.644 1.00 26.09 ATOM 220 CD2 TYR A 31 35.579−9.983 30.121 1.00 29.13 ATOM 221 CE2 TYR A 31 36.616 −9.502 30.870 1.0025.81 ATOM 222 CZ TYR A 31 36.383 −9.009 32.115 1.00 25.77 ATOM 223 OHTYR A 31 37.419 −8.560 32.875 1.00 34.26 ATOM 224 C TYR A 31 31.678−10.274 27.840 1.00 29.45 ATOM 225 O TYR A 31 30.468 −10.172 28.112 1.0030.08 ATOM 226 N GLY A 32 32.141 −10.990 26.808 1.00 29.62 ATOM 227 CAGLY A 32 31.228 −11.746 25.972 1.00 33.28 ATOM 228 C GLY A 32 30.235−10.912 25.198 1.00 36.13 ATOM 229 O GLY A 32 29.161 −11.380 24.832 1.0032.23 ATOM 230 N SER A 33 30.606 −9.668 24.937 1.00 41.08 ATOM 231 CASER A 33 29.722 −8.787 24.217 1.00 47.10 ATOM 232 CB SER A 33 30.130−7.331 24.386 1.00 46.73 ATOM 233 OG SER A 33 29.397 −6.557 23.463 1.0052.64 ATOM 234 C SER A 33 29.816 −9.142 22.764 1.00 57.93 ATOM 235 O SERA 33 30.807 −9.735 22.317 1.00 57.01 ATOM 236 N SER A 34 28.772 −8.75522.039 1.00 65.04 ATOM 237 CA SER A 34 28.657 −8.989 20.613 1.00 70.41ATOM 238 CB SER A 34 28.414 −7.659 19.899 1.00 72.65 ATOM 239 OG SER A34 27.049 −7.299 19.995 1.00 72.85 ATOM 240 C SER A 34 29.885 −9.67120.028 1.00 71.58 ATOM 241 O SER A 34 30.642 −9.053 19.289 1.00 69.77ATOM 242 CB THR A 41 30.810 6.812 19.043 1.00 59.11 ATOM 243 OG1 THR A41 29.666 5.952 18.975 1.00 64.04 ATOM 244 CG2 THR A 41 31.511 6.89217.700 1.00 59.18 ATOM 245 C THR A 41 31.044 6.416 21.449 1.00 51.54ATOM 246 O THR A 41 30.689 5.415 22.079 1.00 55.44 ATOM 247 N THR A 4132.206 4.887 19.817 1.00 58.60 ATOM 248 CA THR A 41 31.763 6.289 20.1051.00 57.60 ATOM 249 N GLY A 42 30.804 7.654 21.870 1.00 35.08 ATOM 250CA GLY A 42 30.159 7.853 23.151 1.00 21.75 ATOM 251 C GLY A 42 29.4099.184 23.225 1.00 18.36 ATOM 252 O GLY A 42 29.011 9.724 22.200 1.0021.32 ATOM 253 N ILE A 43 29.298 9.708 24.431 1.00 21.07 ATOM 254 CA ILEA 43 28.475 10.899 24.619 1.00 22.17 ATOM 255 CB ILE A 43 28.247 11.11326.148 1.00 18.01 ATOM 256 CG2 ILE A 43 29.474 11.764 26.819 1.00 20.05ATOM 257 CG1 ILE A 43 27.124 12.102 26.413 1.00 20.02 ATOM 258 CD1 ILE A43 26.793 12.170 27.882 1.00 22.13 ATOM 259 C ILE A 43 28.947 12.14523.921 1.00 25.61 ATOM 260 O ILE A 43 28.118 12.955 23.466 1.00 18.04ATOM 261 N VAL A 44 30.260 12.336 23.748 1.00 19.35 ATOM 262 CA VAL A 4430.629 13.560 23.090 1.00 19.83 ATOM 263 CB VAL A 44 32.187 13.80923.157 1.00 22.51 ATOM 264 CG1 VAL A 44 32.587 14.916 22.238 1.00 29.90ATOM 265 CG2 VAL A 44 32.568 14.056 24.605 1.00 22.31 ATOM 266 C VAL A44 30.137 13.492 21.655 1.00 18.37 ATOM 267 O VAL A 44 29.664 14.47721.127 1.00 20.08 ATOM 268 N ASP A 45 30.257 12.308 21.027 1.00 19.78ATOM 269 CA ASP A 45 29.821 12.142 19.657 1.00 22.36 ATOM 270 CB ASP A45 30.132 10.738 19.207 1.00 29.90 ATOM 271 CG ASP A 45 31.588 10.39519.435 1.00 38.30 ATOM 272 OD1 ASP A 45 32.374 10.700 18.516 1.00 34.84ATOM 273 OD2 ASP A 45 31.929 9.881 20.546 1.00 39.04 ATOM 274 C ASP A 4528.304 12.345 19.531 1.00 23.03 ATOM 275 O ASP A 45 27.830 13.023 18.6131.00 22.16 ATOM 276 N GLU A 46 27.610 11.772 20.489 1.00 21.13 ATOM 277CA GLU A 46 26.139 11.784 20.505 1.00 24.96 ATOM 278 CB GLU A 46 25.67610.660 21.418 1.00 32.84 ATOM 279 CG GLU A 46 24.220 10.314 21.307 1.0038.09 ATOM 280 CD GLU A 46 23.877 8.973 21.913 1.00 41.51 ATOM 281 OE1GLU A 46 24.713 8.378 22.621 1.00 41.76 ATOM 282 OE2 GLU A 46 22.7398.505 21.689 1.00 45.60 ATON 283 C GLU A 46 25.472 13.084 20.952 1.0024.92 ATON 284 O GLU A 46 24.450 13.450 20.376 1.00 23.35 ATON 285 N CYSA 47 26.045 13.747 21.970 1.00 20.20 ATOM 286 CA CYS A 47 25.477 14.96122.590 1.00 19.56 ATOM 287 C CYS A 47 26.174 16.282 22.474 1.00 28.23ATOM 288 O CYS A 47 25.563 17.328 22.769 1.00 21.99 ATOM 289 CB CYS A 4725.240 14.687 24.091 1.00 21.60 ATOM 290 SG CYS A 47 24.245 13.18624.373 1.00 26.79 ATOM 291 N CYS A 48 27.452 16.263 22.074 1.00 18.45ATOM 292 CA CYS A 48 28.181 17.537 21.940 1.00 17.21 ATOM 293 C CYS A 4828.382 17.818 20.473 1.00 19.88 ATOM 294 O CYS A 48 28.082 18.890 20.0401.00 23.54 ATOM 295 CB CYS A 48 29.521 17.456 22.686 1.00 22.56 ATOM 296SG CYS A 48 30.678 18.814 22.279 1.00 25.89 ATOM 297 N PHE A 49 28.85416.830 19.706 1.00 19.44 ATOM 298 CA PHE A 49 29.044 17.010 18.279 1.0026.47 ATOM 299 CB PHE A 49 29.939 15.912 17.710 1.00 27.72 ATOM 300 CGPHE A 49 31.343 15.940 18.268 1.00 32.07 ATOM 301 CD1 PHE A 49 32.09814.764 18.363 1.00 31.76 ATOM 302 CD2 PHE A 49 31.902 17.133 18.703 1.0029.85 ATOM 303 CE1 PHE A 49 33.396 14.798 18.889 1.00 32.64 ATOM 304 CE2PHE A 49 33.203 17.153 19.230 1.00 29.98 ATOM 305 CZ PHE A 49 33.93815.987 19.320 1.00 24.87 ATOM 306 C PHE A 49 27.706 17.000 17.573 1.0027.42 ATOM 307 O PHE A 49 27.561 17.598 16.517 1.00 31.83 ATOM 308 N ARGA 50 26.744 16.298 18.167 1.00 29.59 ATOM 309 CA ARG A 50 25.381 16.21917.640 1.00 26.05 ATOM 310 CB ARG A 50 25.106 14.832 17.157 1.00 23.82ATOM 311 CG ARG A 50 25.916 14.488 15.899 1.00 26.06 ATOM 312 CD ARG A50 25.953 13.031 15.742 1.00 29.51 ATOM 313 NE ARG A 50 26.583 12.75014.479 1.00 39.53 ATOM 314 CZ ARG A 50 27.849 12.386 14.370 1.00 41.06ATOM 315 NH1 ARG A 50 28.603 12.260 15.466 1.00 40.37 ATOM 316 NH2 ARG A50 28.354 12.198 13.163 1.00 40.98 ATOM 317 C ARG A 50 24.434 16.53918.794 1.00 25.89 ATOM 318 O ARG A 50 24.864 16.563 19.966 1.00 19.42ATOM 319 N SER A 51 23.161 16.811 18.501 1.00 25.83 ATOM 320 CA SER A 5122.229 17.080 19.633 1.00 28.49 ATOM 321 CB SER A 51 21.122 18.08219.248 1.00 29.44 ATOM 322 OG SER A 51 20.679 17.787 17.954 1.00 42.62ATOM 323 C SER A 51 21.569 15.761 20.025 1.00 22.34 ATOM 324 O SER A 5121.229 14.963 19.164 1.00 28.05 ATOM 325 N CYS A 52 21.385 15.517 21.3251.00 21.96 ATOM 326 CA CYS A 52 20.771 14.278 21.762 1.00 24.73 ATOM 327C CYS A 52 19.637 14.683 22.708 1.00 25.51 ATOM 328 O CYS A 52 19.61615.780 23.240 1.00 23.80 ATOM 329 CB CYS A 52 21.791 13.373 22.494 1.0024.55 ATOM 330 SG CYS A 52 22.331 13.922 24.151 1.00 28.05 ATOM 331 NASP A 53 18.671 13.815 22.896 1.00 27.35 ATOM 332 CA ASP A 53 17.62214.228 23.789 1.00 28.62 ATOM 333 CB ASP A 53 16.248 13.872 23.201 1.0034.84 ATOM 334 CG ASP A 53 16.078 12.408 22.968 1.00 39.80 ATOM 335 OD1ASP A 53 16.598 11.616 23.782 1.00 44.65 ATOM 336 OD2 ASP A 53 15.41312.052 21.965 1.00 46.35 ATOM 337 C ASP A 53 17.848 13.616 25.147 1.0023.60 ATOM 338 O ASP A 53 18.735 12.762 25.324 1.00 21.40 ATOM 339 N LEUA 54 17.012 14.005 26.099 1.00 20.38 ATOM 340 CA LEU A 54 17.163 13.55827.477 1.00 14.25 ATOM 341 CB LEU A 54 16.015 14.169 28.329 1.00 21.53ATOM 342 CG LEU A 54 16.024 13.807 29.805 1.00 22.22 ATOM 343 CD1 LEU A54 17.283 14.383 30.476 1.00 21.85 ATOM 344 CD2 LEU A 54 14.733 14.33630.497 1.00 20.36 ATOM 345 C LEU A 54 17.276 12.046 27.683 1.00 23.79ATOM 346 O LEU A 54 18.075 11.598 28.496 1.00 23.74 ATOM 347 N ARG A 5516.449 11.253 26.978 1.00 23.24 ATOM 348 CA ARG A 55 16.489 9.795 27.1041.00 26.67 ATOM 349 CB ARG A 55 15.469 9.140 26.140 1.00 33.74 ATOM 350CG ARG A 55 14.013 9.234 26.618 1.00 47.08 ATOM 351 CD ARG A 55 13.8388.500 27.974 1.00 59.43 ATOM 352 NE ARG A 55 13.580 9.398 29.104 1.0068.64 ATOM 353 CZ ARG A 55 14.359 9.527 30.182 1.00 73.73 ATOM 354 NH1ARG A 55 15.485 8.813 30.302 1.00 74.93 ATOM 355 NH2 ARG A 55 14.02810.401 31.134 1.00 72.23 ATOM 356 C ARG A 55 17.876 9.229 26.774 1.0025.72 ATOM 357 O ARG A 55 18.365 8.348 27.470 1.00 28.77 ATOM 358 N ARGA 56 18.482 9.765 25.726 1.00 21.19 ATOM 359 CA ARG A 56 19.785 9.28525.276 1.00 27.70 ATOM 360 CB ARG A 56 20.049 9.798 23.892 1.00 35.27ATOM 361 CG ARG A 56 19.140 9.134 22.892 1.00 42.72 ATOM 362 CD ARG A 5619.352 7.624 22.897 1.00 52.32 ATOM 363 NE ARG A 56 20.565 7.280 22.1501.00 59.61 ATOM 364 CZ ARG A 56 21.142 6.075 22.108 1.00 61.66 ATOM 365NH1 ARG A 56 20.646 5.046 22.789 1.00 64.42 ATOM 366 NH2 ARG A 56 22.2445.906 21.389 1.00 62.77 ATOM 367 C ARG A 56 20.850 9.724 26.246 1.0025.14 ATOM 368 O ARG A 56 21.755 8.952 26.618 1.00 25.99 ATOM 369 N LEUA 57 20.743 10.967 26.689 1.00 21.87 ATOM 370 CA LEU A 57 21.684 11.44827.701 1.00 22.79 ATOM 371 CB LEU A 57 21.367 12.923 28.059 1.00 22.01ATOM 372 CG LEU A 57 22.188 13.715 29.089 1.00 22.86 ATOM 373 CD1 LEU A57 21.537 15.099 29.190 1.00 27.83 ATOM 374 CD2 LEU A 57 22.115 13.12030.453 1.00 32.64 ATOM 375 C LEU A 57 21.665 10.578 28.970 1.00 20.53ATOM 376 O LEU A 57 22.720 10.208 29.525 1.00 19.79 ATOM 377 N GLU A 5820.486 10.199 29.479 1.00 18.07 ATOM 378 CA GLU A 58 20.425 9.390 30.6821.00 17.97 ATOM 379 CB GLU A 58 18.951 9.351 31.198 1.00 24.34 ATOM 380CG GLU A 58 18.883 8.941 32.660 1.00 36.31 ATOM 381 CD GLU A 58 17.4739.016 33.227 1.00 41.86 ATOM 382 OE1 GLU A 58 17.316 8.902 34.474 1.0039.29 ATOM 383 OE2 GLU A 58 16.549 9.198 32.406 1.00 36.76 ATOM 384 CGLU A 58 21.005 7.942 30.537 1.00 14.19 ATOM 385 O GLU A 58 21.331 7.27931.508 1.00 26.60 ATOM 386 N MET A 59 21.172 7.487 29.307 1.00 15.83ATOM 387 CA MET A 59 21.767 6.166 29.115 1.00 21.59 ATOM 388 CB MET A 5921.626 5.746 27.672 1.00 23.71 ATOM 389 CG MET A 59 20.195 5.145 27.3721.00 27.46 ATOM 390 SD MET A 59 19.916 5.067 25.648 1.00 38.20 ATOM 391CE MET A 59 18.000 5.126 25.597 1.00 38.66 ATOM 392 C MET A 59 23.2616.169 29.521 1.00 23.34 ATOM 393 O MET A 59 23.859 5.124 29.663 1.0025.38 ATOM 394 N TYR A 60 23.847 7.353 29.726 1.00 19.16 ATOM 395 CA TYRA 60 25.266 7.386 30.144 1.00 18.17 ATOM 396 CB TYR A 60 25.982 8.54929.452 1.00 18.20 ATOM 397 CG TYR A 60 26.144 8.364 27.992 1.00 20.05ATOM 398 CD1 TYR A 60 25.193 8.855 27.086 1.00 20.40 ATOM 399 CE1 TYR A60 25.339 8.675 25.713 1.00 22.99 ATOM 400 CD2 TYR A 60 27.245 7.68627.482 1.00 23.58 ATOM 401 CE2 TYR A 60 27.398 7.501 26.131 1.00 24.88ATOM 402 CZ TYR A 60 26.475 7.980 25.258 1.00 25.27 ATOM 403 OH TYR A 6026.676 7.780 23.940 1.00 24.52 ATOM 404 C TYR A 60 25.406 7.424 31.6341.00 22.93 ATOM 405 O TYR A 60 26.519 7.507 32.199 1.00 22.51 ATOM 406 NCYS A 61 24.290 7.385 32.352 1.00 18.42 ATOM 407 CA CYS A 61 24.4027.303 33.809 1.00 15.63 ATOM 408 C CYS A 61 24.808 5.872 34.207 1.0019.70 ATOM 409 O CYS A 61 24.394 4.908 33.563 1.00 25.04 ATOM 410 CB CYSA 61 23.065 7.562 34.511 1.00 22.45 ATOM 411 SG CYS A 61 22.415 9.18134.212 1.00 24.27 ATOM 412 N ALA A 62 25.543 5.738 35.298 1.00 24.74ATOM 413 CA ALA A 62 26.004 4.424 35.756 1.00 31.14 ATOM 414 CB ALA A 6227.273 4.588 36.641 1.00 24.85 ATOM 415 C ALA A 62 24.902 3.747 36.5631.00 36.05 ATOM 416 O ALA A 62 23.920 4.394 36.962 1.00 33.02 ATOM 417 NPRO A 63 25.014 2.427 36.780 1.00 44.06 ATOM 418 CD PRO A 63 26.0211.447 36.310 1.00 47.05 ATOM 419 CA PRO A 63 23.942 1.803 37.576 1.0046.88 ATOM 420 CB PRO A 63 24.182 0.296 37.393 1.00 47.68 ATOM 421 CGPRO A 63 25.651 0.166 37.068 1.00 49.88 ATOM 422 C PRO A 63 24.111 2.25339.027 1.00 48.13 ATOM 423 O PRO A 63 23.135 2.412 39.773 1.00 54.36ATOM 424 N LEU A 64 25.379 2.467 39.379 1.00 51.56 ATOM 425 CA LEU A 6425.835 2.896 40.693 1.00 61.37 ATOM 426 CB LEU A 64 26.997 1.994 41.1701.00 63.79 ATOM 427 CG LEU A 64 26.761 0.984 42.312 1.00 60.23 ATOM 428CD1 LEU A 64 26.872 −0.435 41.767 1.00 61.06 ATOM 429 CD2 LEU A 6427.781 1.210 43.447 1.00 58.43 ATOM 430 C LEU A 64 26.327 4.343 40.6071.00 67.33 ATOM 431 O LEU A 64 27.441 4.612 40.132 1.00 71.54 ATOM 432C1 CYC A 1 19.808 21.082 25.297 1.00 29.47 ATOM 433 C6 CYC A 1 19.97419.730 24.564 1.00 31.45 ATOM 434 O21 CYC A 1 21.316 19.561 24.000 1.0030.05 ATOM 435 C5 CYC A 1 19.707 18.570 25.545 1.00 24.70 ATOM 436 C4CYC A 1 18.349 18.647 26.185 1.00 28.97 ATOM 437 C11 CYC A 1 18.06917.503 27.203 1.00 25.55 ATOM 438 C10 CYC A 1 18.915 17.625 28.449 1.0026.59 ATOM 439 C9 CYC A 1 18.774 19.062 29.167 1.00 22.28 ATOM 440 C15CYC A 1 19.668 19.202 30.407 1.00 18.50 ATOM 441 C18 CYC A 1 19.48818.173 31.529 1.00 17.96 ATOM 442 C17 CYC A 1 20.033 18.861 32.738 1.0020.63 ATOM 443 C16 CYC A 1 20.478 20.370 32.210 1.00 19.19 ATOM 444 C43CYC A 1 20.408 21.271 33.357 1.00 17.91 ATOM 445 C55 CYC A 1 21.47620.723 34.438 1.00 26.47 ATOM 446 C56 CYC B 1 21.498 21.442 35.738 1.0031.90 ATOM 447 C57 CYC B 1 22.417 22.653 36.015 1.00 39.26 ATOM 448 N59CYC B 1 22.473 23.332 37.133 1.00 46.97 ATOM 449 C74 CYC B 1 23.44324.507 37.216 1.00 56.76 ATOM 450 C75 CYC B 1 22.897 25.835 36.400 1.0062.01 ATOM 451 C76 CYC B 1 23.920 27.038 36.528 1.00 66.74 ATOM 452 N77CYC B 1 23.715 27.349 37.968 1.00 68.00 ATOM 453 C78 CYC B 1 24.62327.135 39.017 1.00 72.42 ATOM 454 C80 CYC B 1 24.134 27.596 40.383 1.0076.46 ATOM 455 O86 CYC B 1 22.731 28.037 40.272 1.00 73.13 ATOM 456 C81CYC B 1 25.194 28.755 40.862 1.00 85.35 ATOM 457 O87 CYC B 1 25.45129.858 39.876 1.00 86.13 ATOM 458 C82 CYC B 1 24.767 29.384 42.284 1.0092.20 ATOM 459 O88 CYC B 1 23.400 29.939 42.115 1.00 92.22 ATOM 460 C83CYC B 1 25.777 30.529 42.728 1.00 96.59 ATOM 461 O89 CYC B 1 27.12429.924 42.873 1.00 99.38 ATOM 462 C84 CYC B 1 25.395 31.205 44.130 1.0097.23 ATOM 463 O85 CYC B 1 26.318 32.274 44.541 1.00 95.57 ATOM 464 O79CYC B 1 25.765 26.665 38.880 1.00 74.76 ATOM 465 C68 CYC B 1 21.73723.120 38.324 1.00 39.41 ATOM 466 C69 CYC B 1 20.223 23.496 38.137 1.0038.59 ATOM 467 C70 CYC B 1 19.523 23.355 39.421 1.00 38.22 ATOM 468 N71CYC B 1 20.564 22.987 40.353 1.00 50.02 ATOM 469 C72 CYC B 1 20.39922.763 41.607 1.00 55.62 ATOM 470 C90 CYC B 1 21.157 21.536 42.121 1.0057.53 ATOM 471 O96 CYC B 1 20.407 20.319 41.993 1.00 56.55 ATOM 472 C91CYC B 1 21.664 21.940 43.578 1.00 63.18 ATOM 473 O97 CYC B 1 21.60020.757 44.471 1.00 63.88 ATOM 474 C92 CYC B 1 23.186 22.499 43.622 1.0066.58 ATOM 475 O98 CYC B 1 23.971 21.462 44.307 1.00 68.12 ATOM 476 C93CYC B 1 23.135 23.867 44.446 1.00 67.49 ATOM 477 O99 CYC B 1 22.35724.774 43.597 1.00 66.74 ATOM 478 C94 CYC B 1 24.493 24.656 44.683 1.0074.63 ATOM 479 O95 CYC B 1 24.178 25.865 45.490 1.00 80.12 ATOM 480 O73CYC B 1 19.735 23.526 42.349 1.00 63.35 ATOM 481 O58 CYC B 1 23.10622.977 35.125 1.00 40.42 ATOM 482 C54 CYC B 1 20.742 22.893 32.977 1.0018.54 ATOM 483 C14 CYC B 1 19.521 20.648 31.087 1.00 23.61 ATOM 484 C19CYC B 1 17.911 20.963 31.644 1.00 16.19 ATOM 485 C13 CYC B 1 19.81021.781 30.035 1.00 19.97 ATOM 486 O20 CYC B 1 21.223 21.655 29.535 1.0019.41 ATOM 487 C12 CYC B 1 18.975 21.668 28.869 1.00 17.80 ATOM 488 C8CYC B 1 19.095 20.197 28.184 1.00 20.86 ATOM 489 C3 CYC B 1 18.12920.072 26.867 1.00 26.70 ATOM 490 C7 CYC B 1 16.492 20.310 27.375 1.0027.90 ATOM 491 C2 CYC B 1 18.448 21.212 25.824 1.00 28.19 ATOM 492 BR BRC 1 34.062 6.612 30.395 0.50 33.08 ATOM 493 O HOH W 1 19.167 24.79026.224 1.00 26.47 ATOM 494 O HOH W 2 23.897 21.958 31.132 1.00 22.60ATOM 495 O HOH W 3 21.553 23.642 27.485 1.00 29.42 ATOM 496 O HOH W 424.680 19.970 37.794 1.00 27.43 ATOM 497 O HOH W 5 30.918 19.401 40.6761.00 23.25 ATOM 498 O HOH W 6 16.708 17.200 40.896 1.00 28.59 ATOM 499 OHOH W 7 16.516 25.080 41.353 1.00 25.88 ATOM 500 O HOH W 8 18.741 11.41220.841 1.00 29.70 ATOM 501 O HOH W 9 27.307 23.459 25.693 1.00 22.59ATOM 502 O HOH W 10 29.313 −4.298 41.568 1.00 37.18 ATOM 503 O HOH W 1124.485 23.989 32.854 1.00 35.58 ATOM 504 O HOH W 12 17.430 11.457 36.9701.00 26.36 ATOM 505 O HOH W 13 22.518 16.744 15.421 1.00 37.65 ATOM 506O HOH W 14 22.764 17.648 22.807 1.00 29.54 ATOM 507 O HOH W 15 22.91612.486 18.554 1.00 30.85 ATOM 508 O HOH W 16 21.127 13.160 16.233 1.0067.05 ATOM 509 O HOH W 18 33.941 3.332 34.867 1.00 47.13 ATOM 510 O HOHW 19 28.659 24.132 37.361 1.00 34.15 ATOM 511 O HOH W 20 26.049 12.10038.687 1.00 43.38 ATOM 512 O HOH W 22 22.998 5.484 43.119 1.00 56.07ATOM 513 O HOH W 23 21.091 10.590 19.804 1.00 37.65 ATOM 514 O HOH W 2427.553 20.681 37.920 1.00 40.51 ATOM 515 O HOH W 25 21.539 4.646 32.5381.00 35.17 ATOM 516 O HOH W 35 36.200 4.986 34.099 1.00 55.94 ATOM 517 OHOH W 36 25.301 25.430 41.805 1.00 45.43 ATOM 518 O HOH W 37 28.06314.145 40.069 1.00 39.11 ATOM 519 O HOH W 38 20.561 25.768 50.428 1.0038.92 ATOM 520 O HOH W 39 22.841 23.744 25.080 1.00 40.88 ATOM 521 O HOHW 40 16.781 11.726 39.998 1.00 65.62 ATOM 522 O HOH W 41 29.705 23.09440.234 1.00 40.48 ATOM 523 O HOH W 42 18.375 14.116 41.324 1.00 58.97ATOM 524 O HOH W 43 15.538 9.778 20.639 1.00 61.25 ATOM 525 O HOH W 4434.144 2.745 20.168 1.00 48.45 ATOM 526 O HOH W 45 20.621 8.952 42.4291.00 33.04 ATOM 527 O HOH W 46 17.087 6.329 28.850 1.00 32.70 ATOM 528 OHOH W 47 25.800 23.668 39.622 1.00 46.80 ATOM 529 O HOH W 48 16.97827.199 25.066 1.00 44.77 ATOM 530 O HOH W 49 22.764 7.118 24.736 1.0038.06 ATOM 531 O HOH W 50 24.770 22.332 46.924 1.00 48.70 ATOM 532 O HOHW 51 21.688 25.678 41.327 1.00 52.53 ATOM 533 O HOH W 52 19.396 17.92242.396 1.00 53.49 ATOM 534 O HOH W 53 21.375 18.431 46.997 1.00 57.91ATOM 535 O HOH W 54 19.736 20.961 17.193 1.00 46.33 ATOM 536 O HOH W 5530.374 4.915 45.161 1.00 52.15 ATOM 537 O HOH W 56 18.588 26.408 48.4361.00 44.58 ATOM 538 O HOH W 57 23.722 24.504 28.990 1.00 38.53

1. A method of treating a mammal suffering from an agonist disorder,said method comprising administering to said mammal an effective amountof a composition comprising a crystal formed by insulin-like growthfactor-1 (IGF-1) of SEQ ID NO: 1 that diffracts x-ray radiation toproduce a diffraction pattern representing the three-dimensionalstructure of the IGF-1, and has approximately the following cellconstants a=31.831 Å, b=71.055 Å, c=65.995 Å, and a space group ofC2221₁, and α=β=γ, wherein the IGF-1 is biologically active whenresolubilized, wherein said agonist disorder is selected from the groupconsisting of hyperglycemic disorders, obesity, increased mass-to-leanratio, heart dysfunctions, wasting, kidney disorders, lung diseases,neurological disorders, neuromuscular disorders, GH- insufficiency,whole body growth disorders, Turner's syndrome, and immunologicaldisorders.
 2. The method of claim 1 wherein the mammal is human.
 3. Themethod of claim 1 wherein the disorder is diabetes, obesity, a heartdysfunction, AIDS-related wasting, a kidney disorder, a neurologicaldisorder, a whole body growth disorder, or an immunological disorder. 4.The method of claim 1 wherein the hyperglycemic disorder is selectedfrom the group consisting of Type I diabetes, Type II diabetes, severeinsulin-resistance, hyperinsulinemia, hyperlipidemia, insulin-resistantdiabetes, Mendenhall's Syndrone, Werner Syndrome, leprechaunism,lipoatrophic diabetes, and other lipoatrophies.
 5. The method of claim 1wherein the kidney disorder is selected from the group consisting ofacute and chronic renal insufficiency, end-stage chronic renal failure,glomerulonephritis, interstitial nephritis, pyelonephritis,glorneruloselerosis, Kirumeistiel-Wilson Syndrome in a diabetic patient,and kidney failure after kidney transplantation.
 6. The method of claim1 wherein the immunological disorder is selected from the groupconsisting of immunodeficiencies, decreased CD4 counts, decreased immunetolerance, and chemotherapy-induced tissue damage.
 7. The method ofclaim 1 wherein the neurological or neuromuscular disorder is selectedfrom the group consisting of peripheral neuropathy, multiple sclerosis,muscular dystrophy, myotonie dystrophy, and catabolic states associatedwith wasting.
 8. The method of claim 1 wherein the whole body growthdisorder is selected from the group consisting of short stature andLaron's syndrome.